A Highly linear cDNA synthesis reactions. Total RNA was purified from HeLa S3 cells using the miRNeasy Mini Kit. cDNA was prepared in a miScript II reverse transcription reaction using HiSpec Buffer for a range of RNA amounts from 10 pg to 2 ï¿½g. Each cDNA was then used as a template in real-time PCR reactions using a miScript Primer Assay for 3 mature miRNAs (miR-16, miR-20a, and miR-21) and the miScript SYBR Green PCR Kit. This data demonstrates the broad dynamic detection range that is achievable when using the miScript II PCR System. B Synthetic miR-21 was used to generate cDNA using the miScript II Reverse Transcription Kit under both the HiSpec and HiFlex buffering conditions. A range of amounts from 10 copies to 106 copies of this cDNA was used in real-time PCR using the miScript PCR System. Real-time PCR was performed on the Rotor-Gene Q real-time PCR insturment. The resultant CT values decreased linearly with increasing miRNA copy number, indicating sensitive detection from a wide range of template amounts.
RNA can be detected, quantified and manipulated through copying via the use of reverse transcriptases (RTs), which are RNA-dependent DNA polymerases. RTs synthesize a strand of DNA that is complementary to the original RNA template and is referred to as cDNA. This cDNA can then be further amplified through PCR, qPCR, or isothermally using, for example, one-step RT-LAMP. Alternately, the cDNA can be used in library preparation for next generation sequencing.
When synthesizing a protein, DNA is transcribed into mRNA which is then translated into a protein. One difference between eukaryotic and prokaryotic genes is that eukaryotic genes often contain introns which are not coding sequences, in contrast with exons, which are DNA coding sequences. During transcription, intronic RNA is excised from the RNA primary transcript and the remaining pieces of the RNA primary transcript are spliced back together resulting in processed mRNA. The mRNA code is then translated into an amino acid chain that comprises the newly made protein.
Using reverse transcriptase polymerases, DNA can be synthesised from mRNA and total RNA. Thus it is a 'complementary' copy of the mRNA, and is thus called complementary DNA (cDNA). cDNA forms the substrate for the majority of qPCR gene expression experiments.
PCR (Polymerase Chain Reaction) is a revolutionary method developed by Kary B Mullis (awarded Nobel Prize for chemistry in 1993) in the 1983. PCR is based on using the ability of DNA polymerase to synthesize new strand of DNA complementary to the template strand of DNA. This technique is having an impact on many areas of , genetics, recombinant DNA research molecular biology, forensic analysis, , and medical diagnostics.
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