cdna synthesis steps Archives - Gen9 Genetics
Gene expression studies using quantitative reverse transcription PCR (qRT-PCR) can be challenging when attempted with samples from a few cells or from a single cell. Epicenter's Message BOOSTER ™ Cell Lysate cDNA Synthesis Kit enables RNA amplification and cDNA synthesis directly from cell lysates. A single cell Message BOOSTER kit reaction produces enough cDNA for thousands of qPCRs, even with low abundance messages.
Principal
Although qRT-PCR studies provide valuable information on the relative expression of transcripts in a cell, they are difficult to perform with a very small number of cells (eg, 1 to 1000 cells), for various reasons. These include the difficulty in purifying extremely small amounts of total RNA, the lack of sensitivity, the need to collect samples often due to the very limited amount of RNA available, and the inability to archive samples for future analysis. The new MessageBOOSTER cDNA Synthesis from Cell Lysates Kit eliminates these difficulties by allowing RNA amplification and cDNA synthesis directly from cell lysates, without the need to purify total RNA.
Here we demonstrate that a MessageBOOSTER kit reaction produces enough cDNA directly from a single cell lysate for thousands of qPCRs, and that the cDNA produced allows sensitive detection of even low abundance transcripts.
An oligo (dT) primer containing a T7 promoter and MMLV reverse transcriptase is then used to synthesize the first strand cDNA from poly (A) + RNA. Following second strand cDNA synthesis, a high throughput in vitro transcription reaction is used to amplify the poly (A) + RNA. The in vitro transcription reaction generates thousands of RNA molecules for each double-stranded cDNA template molecule and thus serves as a powerful engine to amplify poly (A) + RNA. After in vitro transcription, the samples are treated with DNase I to remove the double-stranded cDNA template, as well as a large part of the genomic DNA. The amplified RNA is purified using a spin column and then reverse transcribed, using random primers, into cDNA.
We designate the cDNA produced by the MessageBOOSTER kit reaction as "MessageBOOSTER cDNA". For control reactions, we synthesized cDNA directly from cell lysates without an RNA amplification step. This cDNA, called a "cell cDNA" control, was prepared by reverse transcription using an oligo (dT) primer in a 20 µl reaction. In some cases, we use a commercial cell cDNA kit. We also ran a MessageBOOSTER kit reaction without the initial reverse transcriptase. This control, designated the 'no RT' control, was performed to assess the level of residual genomic DNA in the samples after the MessageBOOSTER kit reaction.