Reflecting Total Expression with cDNA Libraries
Despite the introduction of highly efficient DNA microarrays, cDNA libraries still rank as a primary method by which researchers study gene expression. In the more than 10 years since cDNA libraries became popular, techniques for constructing libraries have improved, making them better representations of the population of mRNA in a cell.
Constructing a cDNA library can be a lengthy process involving multiple steps. Barring the purchase of pre-made libraries or contracting with a cDNA library construction service, you can purchase kits that speed up the process. In general, construction begins with the isolation of mRNA, whose poly A tail allows for its capture using an oligo dT column. Reverse transcriptase is then used to copy the mRNA into the first strand of cDNA; DNA polymerase makes the second strand. RNAse, which can hamper the process in the early stages, is then added to degrade the remaining mRNA. The final step is inserting the cDNA into plasmid vectors.
Kits and protocols are available to make cDNA libraries from a wide range of cell types with varying amounts of mRNA. For making libraries from single cells or for small amounts of mRNA, you can purchase cDNA library construction kits that are ready for amplification via PCR. Since a large proportion of mRNAs are present in low amounts (of about 15 copies or less), amplification is an important step to ensure that the resulting library includes low abundant transcripts that may represent the genes of interest.
Advances that maximize the transcription of the entire mRNA sequence are also improving the quality of cDNA libraries. Especially challenging has been transcribing mRNAs of 2 kilobases or longer. Now, companies have come up with ways to coax reverse transcriptase to do a complete job of copying the entire 5’ end. Some companies report that their kits can provide full-length cDNAs measuring up to 9 kilobases. And some companies offer kits that circumvent the use of restriction enzymes for vector insertion. This also increases the number of full-length cDNAs, which have internal restriction sites, that are present in the library.
It’s certainly a challenge to produce a mirror image of the approximate 10,000 to 40,000 genes being expressed all at once in a cell. But, with the sophisticated kits on the market—such as those below—you can transform such a daunting task into a seamless daily routine.
well it is one step closer to the migf-1 i know its not there yet but it a move forward at least
