Paraffin embedded tissue direct RT-PCR kit (RNA free extraction)
This reagent kit is free from nucleic acid extraction and can be directly used as an RT-PCR template using sliced lysate. This reagent kit is sufficient to handle 30 sample treatments and 50 RT PCR reactions.
Kit features:
Reverse transcription can flexibly select random primers, Oligo dT primers, or specific primers, suitable for various situations.
PCR mix contains loading dyes, so PCR products can be directly loaded for electrophoresis.
Two tube operation, RT and PCR are carried out step by step, making it easy to optimize the conditions for RT and PCR reactions separately.
High amplification efficiency, capable of amplifying up to 1kb of RNA.
Kit composition:
Component specifications
RNA free FFPE solution A 30mL
RNA free FFPE solution B 18mL
MMLV (including RI) 100 μ L (red cap)
RT Buffer (including dNTP) 250 μ L (white cover)
Oligo (dT) 18 primer (0.5 μ G/uL) 50 μ L (green cover)
Random primer (0.2 μ G/uL) 50 μ L (natural color cover)
RNase free water 1mL (bright yellow cap)
PCR MagicMix 3.0 with dye 1mL (red cap)
Storage condition: -20 ℃, valid for one year.
Usage:
1、 Release of sample RNA
If there are N samples, N+2 extractions can be set, with the additional one being PC (positive control for sample preparation) and NC (negative control for sample preparation). Water can be used as a negative control for preparation, and after extraction, the template DNA can be obtained and placed on ice for later use.
Add 5 pieces with a thickness of 6-8 μ Transfer m paraffin embedded tissue slices to a 1.5mL plastic centrifuge tube (preferably using a spiral cap centrifuge tube) and add 1mL of solution A. Shake at maximum speed on an oscillator for 10 seconds, and then centrifuge.
Take out 0.6mL of the supernatant from the previous step, add 0.6mL of solution B, and shake for 5-10 seconds.
Leave at room temperature for 10 minutes.
The mixture can be directly used as a template for PCR, RT-PCR, LAMP, and RT-LAMP amplification. The unused templates can be stored at -80 ℃ for a long time.
2、 RT reaction synthesis of cDNA
Prepare the RT reaction system (20) according to the following table μ L system):
Ingredient dosage
RNA template total RNA 100-500ng
Or poly (A) mRNA 10-500ng
Or specific RNA (as prepared by in vitro transcription) 0.01pg-500ng
Primer Oligo (dT) 18 (0.5 μ G/ μ L) 1 μ L
Or random primers (0.2 μ G/ μ L) 1 μ L
Alternatively, a self prepared template specific reverse primer (10pmol/ μ L) 1-2 μ L
RNase free water to 12 μ L
Note: RNA samples should not contain genomic DNA contamination. The ratio of random primers to RNA templates will determine the average length of cDNA synthesis (inversely proportional). In general, the efficiency of random primers is better than that of Oligod