Lab product – RNA Extraction
RNAGEM™ Tissue – Superior RT-qPCR from
Cells Populations 1-150,000 cells BY MICHELLE MILES, DUNCAN KAY, DAVID SAUL
INTRODUCTION The use of cell lysates for direct RT-qPCR is requisite in many clinical and biomedical research applications where cellular populations, down to single cells, need to be analyzed in isolation. Such sample formats are not readily prepared by column or precipitation based methods because of limited recovery, potential for contamination, and sample loss. Nor are these samples suited to traditional quantitation methods, such as UV absorbance and fluorescent dyes due to limited sensitivity at the lower levels of sample abundance. In such cases the best solution for quantitation is the use of qPCR with the genomic DNA as a template. RNAGEM™, a new rapid cell lysate preparation kit, delivers superior sensitivity for both gene expression profiling and sample mass normalization using qPCR. The kit makes use of a highly-active thermophilic protease which strips protein from mRNA templates whilst lysing cells and destroying RNAses. The enzymes and reagents used in ZyGEM kits have been selected to be fully compatible with most downstream applications thereby removing the need for stop solutions and overcoming salt imbalances common to chemical lysis or proteinase K methods. The result is an RNA extract that is immediately ready for use in sensitive RT-qPCR assays. RNAGEM™ uses a rapid, single-step protocol that releases RNA and DNA with excellent linearity across a wide range of cell numbers. The Method is automatable, closed-tube and does not require further purification of the RNA for accurate RT-qPCR analysis. The reagents efficiently lyse the cells and strip protein complexes from nucleic acids, thereby allowing higher processivity of polymerases. The result is greater sensitivity - especially with low abundance transcripts. Reduced handling, and efficient template preparation means that the RNAGEM™ kits generate mRNA profiles that are as close to the biological reality of the sample as possible.
Gene Expression Analysis by RT-qPCR and Sample Normalisation by qPCR Gene Expression Analysis by RT-qPCR HeLa cell numbers from 10-50,000 were extracted using RNAGEM™ and a commercial acid-phenol extraction kit. Plots were generated from a high abundance mRNA (ACTB; ß-actin) and a low abundance mRNA (BRCA1; breast cancer early onset). The clean traces with gradients similar to the standards demonstrate the lack of inhibition.
High copy number mRNA (ACTB)
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Biotechnology Focus june 2010
Reply Card #4748
Low copy number mRNA (BRCA1)
