Applications
- Preparation of RNA from cultured adherent and suspension cells for
RT-PCR.
The QuickExtract™ RNA Extraction Kit is a fast, simple way of
preparing RNA for RT-PCR (both end-point and real-time). The single-tube
system requires only vortex mixing to lyse the cells, and prepare the
RNA for cDNA synthesis. The result is easy processing of one to hundreds
of samples in minutes, with no sample loss or toxic organic solvents.
The QuickExtract RNA Extraction Solution works with cultured adherent
and suspension cells including buccal cells, and has been tested on human,
mouse, rat, E. coli, and S. aureus cell cultures. It is
not suitable for tissue samples or plant samples. An optional DNase I
treatment may improve certain downstream applications.
Benefits
- Single-tube system.
- Prepare lysate in minutes.
- No toxic organic solvents.
- Suitable for high-throughput applications.
- Optional DNase I treatment.
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Figure 1. End-point RT-PCR of different regions of a 14 kb message
using HeLa cell extract with the QuickExtract™ RNA Extraction Kit. A
sample containing 105 HeLa cells was lysed in 100 µl
of QuickExtract RNA Extraction Solution by vortex mixing. The lysate
was reverse transcribed with the MMLV Reverse Transcriptase 1st Strand
cDNA Synthesis Kit using standard conditions and random primers. The
cDNA was then amplified with six primer sets to p532 using the FailSafe™ PCR
System. Lane M, 100 bp ladder; Lane 1, 12984 to 13892; lane 2, 9406
to 10202; lane 3, 5194 to 5802; lane 4, 4191 to 4690; lane 5, 2280 to
2676; and lane 6, 1029 to 1329. |
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Figure 2. Comparative yield of RT-PCR product with different RNA extraction
kits. Lysates were prepared according to manufacturers' instructions
and used as template to produce cDNA using the MMLV RT 1st Strand cDNA
Synthesis Kit, followed by PCR using the FailSafe™ PCR System with
primers for the ALDOA gene. Products were separated on a 2% agarose gel
and stained with SYBR® gold. Lane M, 100 bp ladder; lanes
1 and 2, QuickExtract™ RNA Extraction Kit; lanes 3 and 4, kit from
Vendor 1; lanes 5 and 6, kit from Vendor 2. |
