Editing Complementary DNA (section)

==== First-strand synthesis ====
Using a reverse transcriptase enzyme and purified RNA templates, one strand of cDNA is produced (first-strand cDNA synthesis). The M-MLV reverse transcriptase from the Moloney murine leukemia virus is commonly used due to its reduced [[Ribonuclease H|RNase H]] activity suited for transcription of longer RNAs.<ref>{{Citation |last=Haddad |first=Fadia |title=Reverse Transcription of the Ribonucleic Acid: The First Step in RT-PCR Assay |date=2010 |work=RT-PCR Protocols: Second Edition |volume=630 |pages=261–270 |editor-last=King |editor-first=Nicola |series=Methods in Molecular Biology |publisher=Humana Press |language=en |doi=10.1007/978-1-60761-629-0_17 |isbn=978-1-60761-629-0 |pmid=20301003 |last2=Baldwin |first2=Kenneth M.}}</ref> The AMV reverse transcriptase from the avian myeloblastosis virus may also be used for RNA templates with strong secondary structures (i.e. high melting temperature).<ref>{{Cite web |last=Martin |first=Karen |title=Reverse Transcriptase & cDNA Overview & Applications |url=https://www.goldbio.com/articles/article/Reverse-Transcriptase-cDNA-Overview-Applications |access-date=20 May 2020 |website=Gold Biotechnology}}</ref> cDNA is commonly generated from mRNA for gene expression analyses such as [[Real-time polymerase chain reaction|RT-qPCR]] and [[RNA-Seq|RNA-seq]].<ref>{{Cite web |date=10 August 2017 |title=qPCR, Microarrays or RNA Sequencing - What to Choose? |url=https://www.biosistemika.com/blog/qpcr-microarrays-rna-sequencing-choose-one/ |access-date=20 May 2020 |website=BioSistemika |language=en-US}}</ref> mRNA is selectively reverse transcribed using oligo-d[[Thymine|T]] primers that are the reverse complement of the [[Polyadenylation|poly-adenylated]] tail on the 3' end of all mRNA. The oligo-dT primer anneals to the poly-adenylated tail of the mRNA to serve as a binding site for the reverse transcriptase to begin reverse transcription. An optimized mixture of oligo-dT and [[random hexamer]] primers increases the chance of obtaining full-length cDNA while reducing 5' or 3' bias.<ref>{{Cite web |title=cDNA Synthesis {{!}} Bio-Rad |url=https://www.bio-rad.com/featured/en/cdna-synthesis.html |access-date=28 May 2020 |website=www.bio-rad.com}}</ref> [[Ribosomal RNA]] may also be depleted to enrich both mRNA and non-poly-adenylated transcripts such as some [[non-coding RNA]].<ref>{{Cite journal |last=Herbert |first=Zachary T. |last2=Kershner |first2=Jamie P. |last3=Butty |first3=Vincent L. |last4=Thimmapuram |first4=Jyothi |last5=Choudhari |first5=Sulbha |last6=Alekseyev |first6=Yuriy O. |last7=Fan |first7=Jun |last8=Podnar |first8=Jessica W. |last9=Wilcox |first9=Edward |last10=Gipson |first10=Jenny |last11=Gillaspy |first11=Allison |date=15 March 2018 |title=Cross-site comparison of ribosomal depletion kits for Illumina RNAseq library construction |journal=BMC Genomics |volume=19 |issue=1 |pages=199 |doi=10.1186/s12864-018-4585-1 |issn=1471-2164 |pmc=6389247 |pmid=29703133 |doi-access=free}}</ref>