latest news

07/15/09

Welcome to the new version of the real time PCR website. Little information has changed, but the primer list has been updated!

 

Preparing your cDNA

Rather than do reverse transcription real-time PCR (RT/RT PCR), my protocols generate the cDNA ahead of time. The cDNA generation protocol is the standard one from Promega's MLV-RT or Invitrogen's Superscript III.

The Recipe spreadhseet in the toolbar on the left contains recipes for generating cDNA using either of these two methods.  For cell culture based cDNA generation, I prefer to use Promega's MLV-RT, because it is significantly cheaper than Superscript III.  For cDNA generated from animal tissue, I use Invitrogen's Superscript III. 

Typically I will prepare cDNA from 1 ug of RNA. Sometimes animal tissue doesn't provide that much RNA. 200 ng is acceptable. If your concentrations won't allow you to use 1 ug, use as much as you can, but use equal amounts in all samples.

You can use either oligo dT (which converts only mRNA to cDNA) or random hexamers (which converts mRNA and rRNA to cDNA). Random hexamers are better, because they don't bias your set for 3' ends of transcripts.

Occasionally you will have tissue with very small amounts of starting RNA.  In this situation, I use Qiagen's Sensiscript kit, which is expensive, but works very well. 

Finally, for extremely small amounts of starting RNA, you can use RNA amplification kits.  These are extremely expensive (potentially running around $20 per sample).  These kits are often used for microarrays which require large amounts of starting RNA.  I haven't tried any of these kits yet, and cannot make any recommendations. 

A calculator is provided to help you with this.