Culture Collections

Quality Procedures for cDNA Products

ECACC scientists extract RNA using the Promega Simply RNA kit. The RNA is routinely analysed by gel electrophoresis to check integrity and quantified using a Nanodrop spectrophotometer which also generates 260:280 and 260:230 purity data. During the development phase the RNA was also analysed using an Agilent bioanalyser.

 

Agilent Electropherogram

 Gel Electrophoresis For Cdna Quality Page

 

Summary of typical quality scores for RNA prepared by ECACC using ECACC cell lines

 

260:280 ratios

2.05

260:230 ratios

2.1

RIN score

9.875

18S:28S ratio

2.3

 

The presence of genomic DNA carry-over is then checked by using the RNA in a PCR reaction with PCR primers designed to the intronic sequences of a standard, constitutively expressed housekeeping gene.

1µg of RNA is used as template in a first strand cDNA synthesis reaction using the Qiagen Omniscript cDNA synthesis kit. This is primed using an anchored oligo dT primer for maximal synthesis of gene sequences. The presence of cDNA is then confirmed by using the cDNA as a template in a PCR reaction using PCR primers which detect human cDNA sequences only and do not co-amplify processed pseudogenes. The results of this analysis during the development of the cDNA products is shown below.

 

Agaorose Gel Electrocphoresis For Cdna Quality Page 

 

ECACC supply cDNA as a 20µl aliquot which is routinely diluted 1:1 with water during its characterisation programme. 1µg of RNA (assuming 5% mRNA content) would be expected to generate approximately 30 – 35ng of cDNA assuming a reverse transcription efficiency of 60 – 70%, although as this is a cell culture derived product it cannot be guaranteed.

cDNA Product Introduction Page

Frequently Asked Questions on cDNA

 

 

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