6 minute read
Lab Product – Reagents
RNAGEm™ Tissue – superior rT-qpCr from Cells populations 1-150,000 cells
BY MICHELLE MILES, DUNCAN KAY, DAVID SAUL
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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. Reply Card #4748
High copy number mRNA (ACTB) Low copy number mRNA (BRCA1)
RNAgeM™ extraction Procedure
untreated heLa cells were trypsinised in a canted-neck flask, and resuspended in PBS. The cells were counted physically and a dilution series set up for the extractions. Aliquots from 10 – 50,000 cells were added to the followed extraction volumes:
Cells Volume RNAGEM™ Incubation time
50,000-100,000 50 μl 1 μl 5 min 5000-40,000 25 ul 1 μl 5 min 100-5000 10 ul 0.5 μl 5 min <100 5 ul 0.2 μl 5 min
note: When using standard overnight cultures a single volume and regent ratio can be determined for the growth conditions and range of cellular yields from an given cell-line and growth conditions
The heLa cell dilutions were added to the wells containing the RNAGEm™ reagents with a double aspiration and dispense to ensure mixing.
The samples were then subject to a 75°C incubation for 5 minutes, before being ramped to 4°C.
sample normalisation by qPcR A prerequisite for using gdnA for sample normalisations is that the extraction efficiency of both dnA and rnA is consistent over the range of cell numbers likely to be encountered. Furthermore, this consistency must apply to both low and high copy mrnAs.
To demonstrate RNAGEm™ ability to produce rnA and dnA in consistent, linear proportions, 10-10000 heLa cells were extracted using RNAGEm™ Tissue. The mrnA was amplified by rT-qpCr using qscript cdnAsupermix. dnA was amplified using qpCr with perfeCTA® SYBR® Green FastMix®, ROx™ (Quanta Biosciences).
Copies of the human GApdh gene detected by qpCr plotted alongside copies detected by RT-qPCR of three mRNAs. ACTB = 366 copies per cell; GAPDH = 2607 copies per cell; BRCA1 26 copies per cell.
RNAGEM Tissue
temperatur e
100 90 80 70 60 50 40 30 20 10 0 A dd reagents to cells Easily automated
Hands - off / Closed - tube Hands off/Closed tube - off / Closed - tube
Incubate in a thermal cycler
RNAGEM activated. • Cells lyse, • Ribonucleases destroyed • Nucleoprotein removed DNA/RNA read y
5 10
15
RNAGEM Tissue PLUS
temperatur e
100 90 80 70 60 50 40 30 20 10 0 A dd reagents to cells Easily automated
Hands - off / Closed - tube Hands- off/Closed -tubeHands - off / Closed - tub Hands - off / Closed - tube Hands off/Closed tubes - off / Closed - tube
RNAGEM activated. • Cells lyse, • Ribonucleases destroyed • Nucleoprotein removed Incubate in a thermal cycler
A dd DNAse + buff er RNAGEM inactive. DNAse active RNA
RNAGEM activated.
DNAse destroyed
5 10
15
conclusions • One mix & one step – closed tube protocol. No danger of contamination, sample loss, or sample skew. • Excellent linearity shown for both qPCR and RT-qPCR across a wide range of cell numbers • The protocol yields excellent linearity of yield for both RNA and dnA as determined by functional quantitative pCr assays. • The technology enables the sensitive and reproducible profiling of low abundance transcripts in small cellular populations. • RNAGEm™ offer the ability to significantly reduce costs in time and reagents usage for many functional cell culture assays, such as: • gene silencing experiments • compound screening experiments • RNAGEm™ allows reliable and reproducible rT-qpCr results from overnight cultures and limited cell populations
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