Typical cDNA Synthesis Protocol;
The PCR technique can be applied only to DNA strands. However, with the help of reverse transcriptase, RNA can be transcribed into DNA, thus making PCR analysis of RNA molecules possible. There are two strategies that combine reverse transcription and PCR: one-step RT-PCR and two-step RT-PCR. If the RT step is performed in the same tube with PCR, the process is called one-step PCR. When the PCR and RT are performed in separate tubes, the process is called two-step PCR. With either method, one can usually obtain sufficient cDNA from 1 µg total RNA.
SuperScript ® Double-Stranded cDNA Synthesis Kit - …
Objective: to make complementary (cDNA) from total RNA. Note that if you are planning to make cDNA for QPCR or RT-PCR, the RNA should have already been treated with DNase.
The synthesis of DNA from an RNA template, via reverse transcription, produces complementary DNA (cDNA). Reverse transcriptases (RTs) use an RNA template and a short primer complementary to the 3' end of the RNA to direct the synthesis of the first strand cDNA, which can be used directly as a template for the Polymerase Chain Reaction (PCR). This combination of reverse transcription and PCR (RT-PCR) allows the detection of low abundance RNAs in a sample, and production of the corresponding cDNA, thereby facilitating the cloning of low copy genes. Alternatively, the first-strand cDNA can be made double-stranded using DNA Polymerase I and DNA Ligase. These reaction products can be used for direct cloning without amplification. In this case, RNase H activity, from either the RT or supplied exogenously, is required.
see the RNA extraction protocol; Procedure
Polumuri (2002 32, 1224-1225) showed that reverse transcriptases without RNase H activity can limit the sensitivity of RT-PCR detection. Polumuri and colleagues tested the effects of RNase H treatment on RT-PCR detection sensitivity using SuperScript II M-MuLV RNase H- RT to amplify 3 genes (NCX1, NCX2, NCX3). Of these 3 targets, one (NCX2) was detected much more readily when an RNase H step was included after the reverse transcription.
Invitrogen SuperScript Double-Stranded cDNA Synthesis Kit
If the RNA template is not degraded after first strand cDNA synthesis, it can bind to the newly synthesized cDNA and restrict the accessibility of primers during subsequent PCR amplification. RNase H-mediated destruction of the template can prevent this problem and improve the sensitivity of RT-PCR analysis. However, an additional RNase H incubation step prolongs the reaction time and incurs additional costs for the often expensive RNase H. The Roche Transcriptor Reverse Transcriptase has endogenous RNase H activity ideal for digesting the original RNA template.
cDNA Methods, Protocols and Troubleshootings
Figure 1: Overview of first strand cDNA synthesis with different types of RT primers.
Panel A: Oligo(dT)n primer (in this case n = 18)
Panel B: Anchored oligo(dT)n primer (in this case n = 18). Reverse transcription starts at the very beginning of the poly(A) tail.
Panel C: Sequence-specific (gene-specific) primer.
Panel D: Random hexamers.
V = A, C, or G
B = C, G, or T
N = A, C, G, or T
cDNA Synthesis using the Invitrogen SuperScript ..
The In-Fusion SMARTer Library Construction protocol can be completed in fewer steps than other library construction methods due to a highly efficient cDNA synthesis and cloning process. Only three enzymes are required to complete the entire protocol, as opposed to the usual 6–8 enzymes required for other methods. The SMART(er) cDNA synthesis protocol is user friendly and straightforward with no adaptor ligation or tailing steps. Your precious RNA is subjected to the to the least possible handling, thereby minimizing the risk of degradation.