FIELD OF INVENTION
The present disclosure, in general, relates to a process for identifying nucleic acid sequences for molecular biology applications, and more particularly for identifying splice variants of circular RNA (circRNA) by rolling circle RT-PCR (Reverse Transcription Polymerase Chain Reaction) method.
BACKGROUND AND PRIOR ARTS
Circular (circ) RNAs (ribonucleic acid) are a large family of noncoding (nc)RNAs expressed ubiquitously in various organisms including humans [1, 2]. Recent high-throughput RNA sequencing (RNA-seq.) analysis have identified a large number of circRNAs whose size can range from <100 nt to several kb [3]. Although some circRNAs can be generated from ncRNAs, the majority of the reported circRNAs are derived from exons of pre-mRNAs by a process called backsplicing [4]. In addition, some circRNAs are also generated from the introns and some with retained introns between exons [5-7]. Due to lack of free ends, circRNAs are resistant to exonuclease activity and show exceptional stability in the cells [8]. Although tens of thousands circRNAs have been identified to-date, only a few of them have been reported to regulate gene expression by sponging miRNAs and RBPs, and by producing peptides [9].
The circRNAs contain a unique backsplice junction sequence that serves as the key for their identification and quantification [1, 2]. The circRNA sequences are bioinformatically predicted considering the intervening exons present in between the backsplice junction coordinate of the circRNA [3, 7]. Multiple circRNAs with same backsplice sites (circRNA splice variants) can be generated from the same gene locus by alternative exon selection during splicing and backsplicing [10, 11]. Recent studies have focused on identifying the expression of circRNA splice variants in few model systems by using high-throughput RNA-seq methodologies [7, 10, 11]. As the function of the circRNA mostly depends on the mature sequence

of circRNAs, sequence difference in the mature sequence could lead to differential regulation of gene expression by circRNA splice variants [8, 9]. Since the level of circRNAs is lower than linear RNAs and the body of the circRNA is same as the counterpart linear RNA, it is expensive, technically challenging, and often error-prone to derive the full-length circRNA sequence from RNA-sequencing (RNA-seq) data.
There exists in the art examples of circRNA sequencing including CN106801050, wherein is disclosed a method of a circular RNA high-throughput sequencing with establishment of library and a kit thereof. The establishing method sequentially comprises the following steps: extracting total RNA from a sample; removing DNA in the sample; detecting and evaluating the quality of the total RNA in the sample and determining that the RNA quality meets a requirement; removing rRNA; removing linear RNA; constructing the circular RNA library for high-throughput sequencing. The method claims to be high in efficiency, stable, low in residual ratio of the rRNA, high in circular RNA detection efficiency, good in data reproducibility, and high in sequencing result verifying success rate, and is especially applicable to an FFPE tissue and other samples with poor RNA quality. However, a multitude of steps as well as the construction of circular RNA library and high throughput sequencing must escalate the cost of performing the analysis.
Another example in the art is CN107058360, which discloses a method for constructing a circular RNA expression vector based on rapid cloning technology and application thereof. The method provides for the construction of an artificially-improved vector capable of being rapidly colonized based on one-step method and completely and efficiently expressing circular RNA and application thereof. Through the adoption of a special circular RNA excessive expression sequence frame, upstream and downstream specific sequences and enzyme cutting sites and the adoption of a method for inserting circular RNA sequences by using a one-step method (rapid cloning method), the circular RNA is efficiently, completely and conveniently colonized and excessively expressed. The method and the vector can be widely applied to expression of various circular RNA, provide convenience

for researching the action mechanism of the circular RNA and provide the basis for achieving targeted therapy and drug screening by taking the circular RNA as an in-vivo gene. However, this technique does not provide any means for identifying different splice variants of circRNA and is restricted to specific circRNA expression at a time.
Yet another example included in CN107400701 discloses an amplifying and sequencing method of ring RNA molecules. The method comprises the following steps: (1) mixing total RNA of a cell with a random primer and raising the temperature and annealing; and (2) adding an isothermal amplification enzyme with a reverse transcription function and a rolling circle amplification function and dNTP for reverse transcription and amplification. According to the method, the ring RNA needs not to be separated. By directly taking total RNA as a template, reverse transcription and rolling circle amplification are performed synchronously, so that the operating flow is simplified, and the detection efficiency is increased; the method is high in sensitivity and the quantity of samples needed are reduced greatly, and the accuracy is increased. However, use of random primer precludes the detection and identification of splice variants of circRNA.
A further example in the art included in CN105176981, provides a DNA (deoxyribonucleic acid) sequence used for circular RNA (ribonucleic acid) expression and a vector containing the DNA sequence used for circular RNA expression. The vector is inserted in a eukaryotic expression vector, a lentiviral vector, an adenovirus vector or a retroviral vector to form a series of special expression vectors for circRNA. The DNA sequence and the corresponding expression vector have the best effects, are simple and convenient to operate and are stable in results and efficient in expression (the circRNA expression level is increased by more than a hundredfold). The sequence and the corresponding vector can be widely applied to expression of various circRNA, provide a powerful research tool for the functions and mechanisms of circRNA and provide theoretical support for research and development of further determination of circRNA molecules as novel markers and disease treatment targets.

There exists another example in the art as disclosed in EP3054017, which provides circRNA transcripts that are correlated with cardiovascular diseases, acute myocardial infarction. The circRNA of the invention therefore serve as biomarkers in the diagnosis of cardiovascular disorders. The invention provides the new nucleic acid molecules as well as diagnostic kits and compositions comprising the nucleic acids. Modulation of the expression of the circRNA of the invention further leads to endothelial cell sprouting, and therefore is applied as a treatment for cardiovascular diseases or pathological angiogenesis. The invention provides therapeutic agents modulating circRNA expression or function. However, the claimed invention only provides circRNA sequences in relation to a particular organ and its diseases.
Another example in the art included in US20180282809 relates to a method of diagnosing a disease of a subject, comprising the step of determining the presence or absence of one or more circular RNA in a sample of a bodily fluid of said subject; wherein the presence or absence of said one or more circRNA is indicative for the disease. In particular, the application relates to a method for diagnosing the neurodegenerative disease, preferably Alzheimer's disease, in a subject comprises the steps of- determining the level of one or more circRNA in a sample of a bodily fluid of said subject; comparing the determined level to a control level of said one or more circRNA; wherein differing levels between the determined and the control level are indicative for the disease. Furthermore, the application relates to means for detecting circRNAs being a biomarker for a neurodegenerative disease and kits and array comprising nucleic acid probes for detecting exon-exon junctions in a head to tail arrangement of these circRNAs. However, the method claimed in said invention is specific for disease detection, particularly such as Alzheimer’s and does not identify splice variants of circRNA.
Yet another example in the art includes CN107723373, which discloses the application of a circular RNA molecular marker related to the activation of the ovary of the honeybee queen. The change of the expression quantity of the circular RNA molecule in the activation process of the ovary represents the activation of the

ovary of the honeybee queen and the species of the honeybee queen. The invention further provides a method for identifying the activation state of the ovary of the honeybee queen and the species of the honeybee queen. The provided molecular marker is of great significance for conducting the molecular breeding and breeding the high-fertility honeybee queen. However, the claimed invention is highly specific for a particular animal class, performing a specific biological manipulation in the animal.
A further example in the art is WO2018210173, which describes circRNA as a breast cancer marker and an application thereof. The circRNA is selected from circTADA2A-E6, circTADA2A-E5/E6, circNOL10, circNSUN2, circCSRNP2, circFAM125B, circCDC27, and circABCC1. The claimed invention also relates to a kit, a microarray, and a medication used for diagnosis, prognosis, and treatment of breast cancer, and related methods and applications. However, just as in the previous cases, the claimed invention is specific to a particular disease, breast cancer, and is used as a biomarker for it.
A still further example in prior art is included in CN107384909, which claims a processing method of gastric cancer circRNA. The method comprises the following steps: acquiring gastric cancer tissues and normal para-carcinoma tissues which are removed from a human body, and freezing the tissues; respectively extracting total RNA of the gastric cancer tissues and the normal para-carcinoma tissues; screening out circRNA and mRNA in the total RNA of the gastric cancer tissues and the normal para-carcinoma tissues, and hybridizing the circRNA and the mRNA with an expression chip; and screening out circRNA and mRNA, which have expression differences, of the gastric cancer tissues and the normal para-carcinoma tissues, and implementing integration analysis on the circRNA and mRNA, which have obvious differences, of the gastric cancer tissues and the normal para-carcinoma tissues. According to the processing method of the gastric cancer circRNA, an expression change rule of circRNA expression disorder in gastric cancer is discovered preliminarily, and occurrence and development molecular mechanisms of the gastric cancer are further expounded; therefore, an enough theoretical basis is

offered for searching an effective gastric cancer early diagnosis and malignant progression biological molecular marker. However, the method claimed in said invention is specific for a disease, in this case gastric cancer and acts as a biomarker.
Accordingly, there is a clear felt need in the art for providing a simple, economical, efficient and sensitive method for the identification of different circRNA splice variants. The present invention meets the long-felt need.
The non-patent literature referenced throughout the complete specification with numerals in parentheses have been listed in the following section under the title of ‘References’.
References
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[2] J. Salzman, C. Gawad, P.L. Wang, N. Lacayo, P.O. Brown, Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types, PLoS One, 7 (2012) e30733.
[3] P. Glazar, P. Papavasileiou, N. Rajewsky, circBase: a database for circular RNAs, RNA, 20 (2014) 1666-1670.
[4] Y. Zhang, W. Xue, X. Li, J. Zhang, S. Chen, J.L. Zhang, L. Yang, L.L. Chen, The Biogenesis of Nascent Circular RNAs, Cell Rep, 15 (2016) 611-624.
[5] Y. Zhang, X.O. Zhang, T. Chen, J.F. Xiang, Q.F. Yin, Y.H. Xing, S. Zhu, L. Yang, L.L. Chen, Circular intronic long noncoding RNAs, Mol Cell, 51 (2013) 792-806.
[6] Z. Li, C. Huang, C. Bao, L. Chen, M. Lin, X. Wang, G. Zhong, B. Yu, W. Hu, L. Dai, P. Zhu, Z. Chang, Q. Wu, Y. Zhao, Y. Jia, P. Xu, H. Liu, G. Shan, Exon-intron circular RNAs regulate transcription in the nucleus, Nat Struct Mol Biol, 22 (2015) 256-264.
[7] A.C. Panda, S. De, I. Grammatikakis, R. Munk, X. Yang, Y. Piao, D.B. Dudekula, K. Abdelmohsen, M. Gorospe, High-purity circular RNA isolation method (RPAD) reveals vast collection of intronic circRNAs, Nucleic Acids Res, 45 (2017) e116.

[8] S. Memczak, M. Jens, A. Elefsinioti, F. Torti, J. Krueger, A. Rybak, L. Maier, S.D. Mackowiak, L.H. Gregersen, M. Munschauer, A. Loewer, U. Ziebold, M. Landthaler, C. Kocks, F. le Noble, N. Rajewsky, Circular RNAs are a large class of animal RNAs with regulatory potency, Nature, 495 (2013) 333-338.
[9] A.C. Panda, I. Grammatikakis, R. Munk, M. Gorospe, K. Abdelmohsen, Emerging roles and context of circular RNAs, Wiley Interdiscip Rev RNA, 8 (2017).
[10] X.O. Zhang, R. Dong, Y. Zhang, J.L. Zhang, Z. Luo, J. Zhang, L.L. Chen, L. Yang, Diverse alternative back-splicing and alternative splicing landscape of circular RNAs, Genome Res, 26 (2016) 1277-1287.
[11] Y. Gao, J. Wang, Y. Zheng, J. Zhang, S. Chen, F. Zhao, Comprehensive identification of internal structure and alternative splicing events in circular RNAs, Nat Commun, 7 (2016) 12060.
[12] D.B. Dudekula, A.C. Panda, I. Grammatikakis, S. De, K. Abdelmohsen, M. Gorospe, CircInteractome: A web tool for exploring circular RNAs and their interacting proteins and microRNAs, RNA Biol, 13 (2016) 34-42.
[13] A.C. Panda, M. Gorospe, Detection and Analysis of Circular RNAs by RT-PCR, Bio Protoc, 8 (2018).
[14] N. Wong, X. Wang, miRDB: an online resource for microRNA target prediction and functional annotations, Nucleic Acids Res, 43 (2015) D146-152.
OBJECTS OF THE INVENTION
An object of this invention is to identify circRNA splice variants with identical
backsplice junction sequence.
Another object of this invention is to propose a process for preparing tandem repeats
of cDNA from circRNAs.
Yet another object of this invention is to design appropriate novel primer-sets for
the identification of full length circRNA splice variants.
Still another object of this invention is to propose an RT-PCR method followed by
sequencing to identify the exact sequence of a circRNA.
A further object is to identify circRNA splice variants with identical backsplice
junction sequence.

Still further object of this invention predicts the differential association of miRNAs with circRNAs for predicting their biological functions.
SUMMARY OF THE INVENTION
The present invention discloses sets of oligonucleotide primers for the accurate identification of circRNA splice variants having identical backsplice junctions. The present invention comprises forward primers uniquely designed to identify and hybridize to the backsplice junction while the reverse primers hybridize to circRNA sequence exactly upstream of the forward primer to amplify full length of target splice variant. The oligonucleotide forward primer span at least 10 nucleotides upstream and downstream of backsplice junction sequence. The oligonucleotide primer pairs sets comprising SEQ ID NO. 1 to SEQ ID No. 6 have been designed based on gene sequences for the splice variants of hsa_circ_0007127, hsa_circ_0007822 and hsa_circ_0007700 in HeLa cells. The designed oligonucleotide primer sets underlying the present invention have also been used to identify circRNAs with altered full-length sequences which is responsible for differential association of with miRNAs.
The present disclosure also provides a method for the identification and amplification of circRNA splice variants with identical backsplice junction comprising of the steps of- a) enrichment of total RNA by digestion with RNAse R followed by synthesis of tandem repeats of cDNA by RNAse H- minus reverse transcriptase (RT); b) hybridization of uniquely designed forward primers to the backsplice junction sequence of target circRNA and that of the reverse primer to a sequence of circRNA exactly upstream of the forward primer and amplification of intervening sequence which amplify novel circRNA splice variants with different full-length sequence; and c) sequencing of PCR products with full length primers for the identification of splice variants of reported circRNAs by the Sanger sequencing technique.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
The illustrated embodiments of the subject matter will be best understood by reference to the drawings, wherein like parts are designated by like numerals throughout. The following description is intended only by way of example, and simply illustrates certain selected embodiments systems and processes that are consistent with the subject matter as claimed herein, wherein:
Figure 1: Shows the schematic representation of biogenesis of circRNA splice variants with identical backsplice sequence.
Figure 2(A): Shows the schematic representation of the design of divergent primers used for the specific detection of circRNAs.
Figure 2(B): Shows a SYBR Gold-stained, 2% agarose gel profile wherein, the RT-PCR products amplified with divergent primers have been resolved.
Figure 2(C): Shows a representative profile PCR product purified and sequenced to confirm backsplice circRNA junction sequences.
Figure 3: Shows a graphical representation of RT-qPCR data showing the resistance of circRNAs to RNase R treatment.
Figure 4(A): Shows a schematic representation of rolling circle reverse transcription of circRNAs and the primer for the PCR amplification of full-length circRNAs.
Figure 4(B): Shows a SYBR Gold-stained, 2% agarose gel profile wherein, the amplified RT-PCR products amplified with full-length PCR primers have been resolved.
Figure 5(A): Shows a schematic representation of the hsa_circ_0007127 splice variants biogenesis from CNOT2 pre-mRNA. The black dotted lines represent splicing while blue dotted lines represent alternative splicing.
Figure 5(B): Shows spliced full-length sequences of splice variants of hsa_circ_0007127. The text color corresponds to the color of the exon box in panel A.

Figure 6(A): Shows a schematic representation of the hsa_circ_0007700 splice variants biogenesis from SOAT1 pre-mRNA. The black dotted lines represent splicing while blue dotted lines represent alternative splicing.
Figure 6(B): Shows spliced full-length sequences of splice variants of hsa_circ_0007700. The text color corresponds to the color of the exon box in panel A.
Figure 7(A): Shows the schematic representation of the hsa_circ_0007822 splice variants biogenesis from KDM1A pre-mRNA. The black dotted lines represent splicing while blue dotted lines represent alternative splicing.
Figure 7(B): Shows spliced full-length sequences of splice variants of hsa_circ_0007822. The text color corresponds to the color of the exon box in panel A.
Figure 8(A): Panel shows miRDB predicted miRNA targets of splice variants of hsa_circ_0007127.
Figure 8(B): Panel shows miRDB predicted miRNA targets of splice variants of hsa_circ_0007700.
DETAILED DESCRIPTION OF THE INVENTION
At the very outset of the detailed description, it may be understood that the ensuing description only illustrates a form of this invention. However, such a form is only exemplary embodiment, and without intending to imply any limitation on the scope of this invention. Accordingly, the description is to be understood as an exemplary embodiment and teaching of invention and not intended to be taken restrictively.
Throughout the description and claims of this specification, the phrases “comprise” and “contain” and variations of them mean “including but not limited to”, and are not intended to exclude other moieties, additives, components, integers or steps. Thus, the singular encompasses the plural unless the context otherwise requires. Wherever there is an indefinite article used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with an aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification including any accompanying claims, abstract and drawings or any parts thereof, or to any novel one, or any novel combination, of the steps of any method or process so disclosed.
The reader's attention is directed to all papers and documents which are filed concurrently with or before this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference. Post filing patents, original peer reviewed research paper may be published.
The following descriptions of embodiments and examples are offered by way of illustration and not by way of limitation.
Unless contraindicated or noted otherwise, throughout this specification, the terms “a” and “an” mean one or more, and the term “or” means and/ or.
Recent high-throughput RNA-seq analyses coupled with novel computational tools have begun to identify the vast abundance of circular RNAs (circRNAs) in various organisms including humans [1-3]. However, only a handful of circRNAs have been shown to be functional. Current method for identification and quantification of circRNAs through circRNA-sequencing, circRNA microarray, RT-PCR, and Northern blot analyses, rely on the backsplice junction sequence of circRNA. The mature sequence of the circRNA after backsplicing may be different in different

cells or tissues due to differential splicing events leading to inclusion or exclusion of exons and/or intron sequences. Divergent primers across the backsplice junction sequence have been used till date, for circRNA identification by RT-PCR while the mature spliced sequence of the circRNA is predicted from the transcriptome data [10-13]. Moreover, divergent primers can recognize more than one circRNAs generated from the same exons of the parental gene and are not unique to specific circRNAs. To overcome these issues, in-house designed primer pairs underlying the present invention have been used for full-length amplification and accurate identification of mature sequences of circRNA.
In our scientific study published previously [7], we discovered circRNA splice variants with same backsplice junction sequence for many circRNAs. We selected three circRNAs hsa_circ_0007127, hsa_circ_0007700, and hsa_circ_0007822 which are highly abundant in the human cervical carcinoma (HeLa) cells as well as being widely expressed in many other cell types including Hs68 and Gm12878 [3, 7]. The rolling-circle amplification of circRNA cDNA followed by PCR with the special primer pairs underlying the present invention could identify the alternative splice variants of circRNAs accurately. Interestingly, amplification of full-length sequences of circRNAs revealed expression of circRNA splice variants with same splice junction in HeLa cells. Altered selection of exon sequences during circRNA biogenesis leads to changes in the mature spliced sequences of circRNAs. As circular RNAs are known to regulate gene expression by acting as decoy for miRNAs, we analyzed their interaction with miRNAs using miRDB web tools [14]. As expected, circRNA splice variants were predicted to interact with different miRNAs (Fig. 8). The rolling circle amplification of circRNA to cDNA followed by PCR with the designed primer pairs underlying the present invention may be used for correct identification of mature full-length sequence of circRNAs which is critical for elucidating their function.
The process of identification and amplification of circRNA splice variants with the use of primer pairs underlying the present, while not intending to limit the scope of the invention, has been elaborated in the following sections with examples:

Example 1
Cell culture and RNA isolation
Human cervical carcinoma HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% pen/strep (Thermo Fisher Scientific). The Hela cells were cultured in a humidified atmospheric air with 5% CO2 at 37°C. Total RNA from the HeLa cells was isolated using the PureLink RNA Mini Kit (Thermo Fisher Scientific) following the manufacturer’s protocol.
CircRNA targets and PCR primers
Few circRNAs with potential splice variants were randomly selected from our previous publication for validation. The divergent (div) primers for circRNA detection and quantification were designed by the CircInteractome web tool (sequences have been shown in Table 1) [12]. The divergent primers can detect the backsplice junction sequence irrespective of the sequence of the circRNA body. The selected circRNAs were then validated for their backsplice junction using regular RT-PCR and also assessed by RT-qPCR to show their resistance to RNaseR treatment when compared to their linear counterpart.



The primers for full-length (fl) circRNA amplification were designed manually by placing the forward primer on the junction sequence spanning 10 nt on either side of the junction and placing the reverse primer exactly upstream to the forward primer. The specificity of the primers was checked with NCBI BLAST web tool. The selected circRNAs were then used to amplify the full length sequence of circRNAs with same junction but with probable different overall sequence giving rise to splice variants. The oligo sequences have been shown in Table 2.



RNase R treatment and cDNA synthesis
Total RNA was extracted from the Hela cells using the Purelink RNA isolation kit (Invitrogen) following the manufacturer's instructions. Briefly, 5 μg of total RNA was digested for 30 min at 37 °C with 1 μl (20 Units/µL) of RNase R enzyme (Epicentre) followed by RNA isolation with PureLink RNA isolation kit. For regular cDNA synthesis for qPCR experiments, reverse transcription (RT) was performed with random hexamers and Maxima reverse transcriptase enzyme (Thermo Fisher Scientific) as described previously [13]. For full-length circRNA cDNA, random primer was used with Maxima RNase H-minus reverse transcriptase (Thermo Fisher Scientific) following manufacturer’s instruction to synthesis cDNA. The cDNA reaction was treated with 1µL (5Units/µL) of RNase H enzyme for 15 minutes at 37 °C to digest the RNAs before inactivating the RT enzyme by heating the reaction at 85°C for 5 minutes.
RT-PCR and quantitative PCR (qPCR) analysis of circRNA
Reverse transcription followed by quantitative polymerase chain reactions (RT-qPCR) were performed using Power Up SYBR Green master mix (Thermo Fisher Scientific), with a cycle setup of 2 min at 95°C and 45 cycles of 2 second at 95°C plus 5 second at 60°C was used for RT-qPCR followed by relative RNA level calculation as described previously [7, 13]. Normal PCR amplification was performed with the full-length circRNA cDNAs and the full-length primers using the DreamTaq Green PCR Master Mix with a cycle setup consisting of 2 min at 95°C and 40 cycles of 5 second at 95°C plus 20 sec at 58°C and 1 min. at 72°C.

The PCR products amplified with divergent primers and full-length primers were resolved on SYBR Gold stained agarose gel for visualization followed by Sanger sequencing of the target PCR products.
CircRNA sequencing and miRNA target prediction
The circRNA PCR products were gel purified using the size select 2% gels (Invitrogen) or using the gel elution kit (Invitrogen) following the manufacturer’s protocol. The PCR products were sequenced with the forward or reverse primer by sanger sequencing method. The identified circRNA backsplice junction and full length sequences were compared with the reported circBase sequences. The circBase and the identified splice variant sequences were used to identify the potential miRNA targets of circRNAs using the custom prediction web tool in miRDB (http://mirdb.org) [14].

EXAMPLE 2
Identification of circRNA backsplice sequences in Hela
For the detection of circRNAs specifically, divergent primers were designed using the CircInteractome website (Fig. 2A) [12]. The PCR products from HeLa cell cDNA using divergent primers were resolved on 2% agarose gels stained with

SYBR-Gold (Fig. 2B), followed by purification and sequencing of the PCR products to confirm the backsplice sequences (Fig. 2C) [13]. For the identification of true circRNAs in Hela cells, selected circRNAs were analysed for RNase R resistance in HeLa cell [13]. As expected, the circRNAs tested showed resistance to RNase R treatment while the linear RNAs like 18S rRNA and ACTB mRNAs were degraded to minimal levels (Fig. 3).
EXAMPLE 3
Identification of full-length sequence of circRNA by rolling circle RT-PCR
For the identification of the full-length sequence of circRNAs, tandem repeats of the full-length circRNA from control (ctrl) and RNase R treated HeLa RNA were generated by performing the rolling circle reverse transcription of circRNAs using RNase H-minus RT (Fig. 4A, left). For full-length PCR amplification of specific circRNAs from the above cDNA, the forward primer was placed on the backsplice site and the reverse primer upstream of that can amplify the full-length circRNAs (Fig. 4A, right). The PCR products amplified from the above cDNA full-length primers revealed the expression of circRNA splice variants in HeLa cells (Fig. 4B). For instance, all the tested circRNAs including hsa_circ_0007127, hsa_circ_0007700, and hsa_circ_0007822 showed the expression of splice variants (marked by *) with or without the expression of circBase reported sequence (marked by arrowhead) (Fig. 4B; Table 3).
EXAMPLE 4
Identification of circRNA splice variants with identical backsplice sequence by RT-PCR
The mature spliced sequence of circRNA may vary due to alternative splicing of exons/introns during the circRNAs biogenesis. For instance, the spliced hsa_circ_0007127 in circBase is reported to be 490 nt generated from the exons 2, 3, and 4 of CNOT2 transcript (NR_037615.1) which could not be detected in our

RT-PCR (Fig. 5A). Interestingly, the sanger sequencing of circRNA full-length PCR products revealed that the hsa_circ_0007127 expresses 2 splice variants, one with 266 nt containing exons 2 and 4 while the other one is of 324 nt containing the exon 2, 4, and part of exon 3 (Fig. 5B). Similarly, the hsa_circ_0007700 reported by circBase is of 716 nt length containing 5 exons while the splice variant identified here contains 5 exons, but partial sequence of one of the exons giving rise to a spliced sequence of 612 nt (Fig. 6). In addition, hsa_circ_0007822 reported by circBase is of 816 nt long with 8 exons while the splice variant contains 7 exons with a length of 756 nt (Fig. 7).
EXAMPLE 5
Differential association of miRNAs with circRNA splice variants
Due to changes in the full-length sequence of circRNAs different isoforms of the same circRNAs alter the binding sites for target miRNAs which can differentially regulate their target gene expression. The miRDB web tool was used to predict the differential association of miRNAs with circRNA splice variants. For example, the short and long splice variant of hsa_circ_0007127 contains target sites for 7 and 9 miRNAs while the circBase sequence has 13 miRNA target sites (Fig. 8A). The identified splice variant of hsa_circ_0007700 lacks the binding sites for many miRNAs which are predicted to target the circBase reported sequence (Fig. 8B).
Now, the crux of the invention is claimed implicitly and explicitly through the following claims.

We Claim:
1. Oligonucleotide primers for the identification and amplification of full length circRNA splice variants comprising, a forward primer designed to identify and hybridize to the backsplice junction while the reverse primer is designed to hybridize to circRNA sequence exactly upstream of the forward primer to amplify full length of target splice variant of said circRNA.
2. The oligonucleotide primer pairs as claimed in claim 1 wherein, they identify and amplify circRNA splice variants with identical backsplice junctions.
3. The oligonucleotide forward primer as claimed in claim 1 wherein, they span at least 10 nucleotides upstream and downstream of backsplice junction sequence.
4. Oligonucleotide primer sets for the identification of full length circRNA splice variants with identical backsplice junctions comprising:

(I) A pair of primers of SEQ ID NO. 1 and SEQ ID No. 2;
(II) Another pair of primers of SEQ ID NO. 3 and SEQ ID No. 4; and
(III) Another pair of primers of SEQ ID No. 5 and SEQ ID No. 6.

5. The oligonucleotide primer sets as claimed in claim 4, wherein said primer pair(s) represent forward oligonucleotide primer and reverse oligonucleotide primer complementary to circRNA.
6. The oligonucleotide primer sets claimed in claim 4, wherein said primer pair(s) of SEQ ID No. 1 and 2 represent forward oligonucleotide primer and reverse

oligonucleotide primer designed based on gene sequence for circRNA splice variant hsa_circ_0007127.
7. The oligonucleotide primer sets claimed in claim 4, wherein said primer pair(s) of SEQ ID No. 3 and 4 represent forward oligonucleotide primer and reverse oligonucleotide primer designed based on gene sequence for circRNA splice variant hsa_circ_0007822.
8. The oligonucleotide primer sets claimed in claim 4 wherein said primer pair(s) of SEQ ID No. 5 and 6 represent forward oligonucleotide primer and reverse oligonucleotide primer designed based on gene sequence for circRNA splice variant hsa_circ_0007700.