Jul 24, 2020
Rat Brain Tissue RNA Extraction/cDNA Synthesis for qPCR
This protocol is a draft, published without a DOI.
- 1University of California, San Diego
- George Lab @ UCSDTech. support email: olgeorge@ucsd.edu
Protocol Citation: Kokila Shankar, Olivier George 2020. Rat Brain Tissue RNA Extraction/cDNA Synthesis for qPCR. protocols.io https://protocols.io/view/rat-brain-tissue-rna-extraction-cdna-synthesis-for-7wdhpa6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 03, 2019
Last Modified: July 24, 2020
Protocol Integer ID: 28325
Abstract
This protocol is to obtain cDNA from rat brain tissue micropunches for downstream qPCR. Briefly, RNA will be extracted from brain tissue and will be converted to cDNA through reverse transcription reaction. Protocols have been adapted from Zymo (https://files.zymoresearch.com/protocols/_r2060_r2061_r2062_r2063_direct-zol_rna_microprep.pdf) and New England Biolabs (https://www.neb.com/protocols/2013/01/23/first-strand-cdna-synthesis-protocols-e6560).
Materials
MATERIALS
ProtoScript II First Strand cDNA Synthesis Kit - 150 rxnsNew England BiolabsCatalog #E6560L
Molecular Biology Grade WaterFisher ScientificCatalog #10154604
Ethanol (95 - 100%), molecular grade
TRIZOL reagentInvitrogen - Thermo FisherCatalog #15596-026
Microcentrifuge tubes (1.5 ml)Denville Scientific Inc.Catalog #C-2170
Direct-zol RNA Microprep KitZymo ResearchCatalog #R2062
STEP MATERIALS
TRIZOL reagentInvitrogen - Thermo FisherCatalog #15596-026
Ethanol (95 - 100%), molecular grade
Molecular Biology Grade WaterFisher ScientificCatalog #10154604
Molecular Biology Grade WaterFisher ScientificCatalog #10154604
Molecular Biology Grade WaterFisher ScientificCatalog #10154604
Ensure all reagents are nuclease-free (not just sterile)
Protocol materials
Ethanol (95 - 100%), molecular grade
Molecular Biology Grade WaterFisher ScientificCatalog #10154604
Molecular Biology Grade WaterFisher ScientificCatalog #10154604
Molecular Biology Grade WaterFisher ScientificCatalog #10154604
Ethanol (95 - 100%), molecular grade
Direct-zol RNA Microprep KitZymo ResearchCatalog #R2062
Microcentrifuge tubes (1.5 ml)Denville Scientific Inc.Catalog #C-2170
TRIZOL reagentInvitrogen - Thermo FisherCatalog #15596-026
ProtoScript II First Strand cDNA Synthesis Kit - 150 rxnsNew England BiolabsCatalog #E6560L
Molecular Biology Grade WaterFisher ScientificCatalog #10154604
TRIZOL reagentInvitrogen - Thermo FisherCatalog #15596-026
TRIZOL reagentInvitrogen - Thermo FisherCatalog #15596-026
Ethanol (95 - 100%), molecular grade
Molecular Biology Grade WaterFisher ScientificCatalog #10154604
Molecular Biology Grade WaterFisher ScientificCatalog #10154604
Molecular Biology Grade WaterFisher ScientificCatalog #10154604
Safety warnings
Wear lab coat, gloves, and goggles as the protocol involves use of Trizol. Change gloves frequently to avoid contamination.
Before start
Make sure all surfaces/tools/equipment are sterile and/or have been cleaned with RNase decontaminant spray. Make sure centrifuge has been pre-cooled to 4C. Thaw tissue samples on ice right before starting protocol.
Remove tissue samples from -80 °C and thaw on ice
If samples are stored in RNALater ICE, remove from tube and add 300 µL
TRIZOL reagentBio-rad LaboratoriesCatalog #15596-026
to each sample. Pipet homogenize until tissue is fully dissolved and leave at RT while working on other samples. Spin samples at 13000 RPM at 4 °C for 00:05:00 and transfer supernatant to new tube.
Note
If samples were stored directly in Trizol, ensure tissue is fully dissolved in Trizol, and spin down as directed.
Add 300 µL
Ethanol (95 - 100%), molecular gradeBio-rad Laboratories
to each sample, mix well (invert tube, do not pipet), and transfer mixture to spin columns (with collection tube underneath). Spin samples at 13000 RPM at room temperature (21 °C ) for 00:00:30 and transfer column to new collection tube.
Add 400 µL Direct-zol RNA Prewash buffer to each column. Spin samples at 13000 RPM at room temp (21 °C ) for 00:00:30 and discard flowthrough. Repeat this step (2 total washes).
Add 700 µL RNA Wash buffer to each column. Spin at 13000 RPM at room temp (21 °C ) for 00:02:00 . Spin one more time for 00:01:00 to ensure all liquid is removed from column.
Add 8 µL
Molecular Biology Grade WaterBio-rad LaboratoriesCatalog #10154604
directly to column matrix (be careful not to pierce column) and spin at 13000 RPM at room temp (21 °C ) for 00:00:30 . IMMEDIATELY TRANSFER COLUMNS TO ICE. Quanitify RNA concentration and purity (260/280) using
Equipment
Nanodrop 2000C
NAME
Thermo Scientific
BRAND
TSC-ND2000C
SKU
Note
Make sure all RNA samples stay on ice and as sterile as possible to avoid RNase contamination. Move immediately to next step to limit RNA degradation.
On ice, in separate 200 uL PCR strip tubes, mix up to 1 µg RNA (up to 6 µL ) with 2 µL d(T)23VN and
Molecular Biology Grade WaterBio-rad LaboratoriesCatalog #10154604
(if needed) to make an 8 µL total reaction. Mix samples well and make sure nothing is sticking to the sides of the tube. Make 1 additional tube to serve as negative control.
Note
Master mix of d(T)23VN and/or H2O should be made to avoid pipetting in and out of stock tube
Denature RNA at 70 °C for 00:05:00 and cool down at 4 °C for 00:05:00 . Use preset "cDNAincubate" function on
Equipment
C1000 Touch Thermal Cycler
NAME
PCR machine
TYPE
BioRad
BRAND
1851148
SKU
. Immediately put on ice.
To each sample tube, add 10 µL M-MuLV Reaction Mix and 2 µL M-MuLV Enzyme Mix. To the negative control tube, add 10 µL M-MuLV Reaction Mix and 2 µL
Molecular Biology Grade WaterBio-rad LaboratoriesCatalog #10154604
. Total reaction volume for all samples should be 20 µL .
Note
Ensure this step is done on ice
Incubate samples at 42 °C for 01:00:00 and then inactivate enzyme at 80 °C for 00:05:00 . Use preset "NEB cDNA" program on
Equipment
C1000 Touch Thermal Cycler
NAME
PCR machine
TYPE
BioRad
BRAND
1851148
SKU
Note
At this point, samples can either be used directly in qPCR assay or stored for later use at -20 °C