Reverse Transcription/cDNA Synthesis
Description
The Genome Core performs reverse transcription on total RNA samples using the Bio-Rad iScript cDNA Synthesis Kit. This kit uses both oligo (dT) and random hexamer primers, which enables it to work with a variety of targets. We have used this kit successfully for several years, but we will also be testing several new cDNA kits after the New Year.
Science
The iScript reverse transcriptase is an RNaseH+ modified, MMLV-derived enzyme mixed with an RNase inhibitor. The H+ aspect of the RNase allows greater sensitivity in detecting RNA transcripts and, therefore, less RNA (as little as 100 fg according to manufacturer). The iScript kit uses a blend of oligo (dT) and random hexamers to prime cDNA synthesis which is the recommended method of cDNA synthesis for QPCR.
Protocol
Available here.
Pricing
See pricing schedule here.
What to Provide
Please provide the core with the RNA samples to be reverse transcribed in labeled 1.5ml eppendorf tubes. If you know the concentration of your samples please make note on your order form and provide a MIMIMUM of 1500ng per sample, if possible. If you do not know the concentration please include extra volume for use with our Nanodrop. If we are provided with a limited quantity of RNA we will reverse transcribe the maximum quantity of cDNA possible with the particular sample. Please also remember to upload the appropriate order form on CoreAdmin.
