DNase and Reverse Transcription

DNase treatment 

Please complete and attach the qPCR full service form even if only DNase or RT is required and upload to MyCores when placing the service order.

Description

DNase treatment is performed before cDNA synthesis to remove any genomic DNA from the RNA. This step is essential when using Sybr Green RT=PCR chemistry, which will detect all dsDNA including non-specific products. DNase treatment is also necessary when looking at gene expression levels with primers and probes that do not cross an exon/exon junction.

Reagents

The Genome Core uses Promega RQ1 RNase-Free DNase. Product information can be found here.

Sample requirements

Please provide the core with sufficient volume/concentration of RNA, in particular if downstream RT and QPCR services are requested. One DNase reaction can have a maximum of 8ul of RNA. RNA of low concentration may require multiple DNase treatments to generate sufficient RNA and will be charged accordingly.

 

Reverse Transcription

Description

Reverse transcription is the process in which cDNA is transcribed from RNA. DNAse treatment is sometimes done beforehand to remove any genomic DNA from the RNA. This is especially necessary when looking at gene expression levels with primers and probes that are within a single exon.

Reagents

The Genome Core uses Quanta Biosciences qScript cDNA Synthesis Kit. Further details can be found here.

Sample requirements

Please provide the core with sufficient volume/concentration of RNA, in particular if downstream QPCR services are requested. If known, please note the sample concentration on your order form, we prefer a minimum of 1000 ng/ul if possible. If the sample is of a low concentration include extra volume so we can perform multiple RT reactions if necessary.