Aug 17, 2020

Public workspaceCell harvest and RNA prep using Qiagen RNAeasy Kit

This protocol is a draft, published without a DOI.
  • 1RIKEN
Katrin Blondrath
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Protocol Citation: Katrin Blondrath 2020. Cell harvest and RNA prep using Qiagen RNAeasy Kit. protocols.io https://protocols.io/view/cell-harvest-and-rna-prep-using-qiagen-rnaeasy-kit-bf4sjqwe
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: May 07, 2020
Last Modified: August 17, 2020
Protocol Integer ID: 36722
Materials
MATERIALS
ReagentRNAeasy mini kitQiagenCatalog #74106
ReagentParafilm
Reagentbeta mercaptoethanol
Reagentlow bind nuclease free eppendorf tubes
ReagentRNAse zap
ReagentIce
ReagentEthanol
ReagentNano drop or similar
Reagent384well plate
Reagentseal for 384well plate
ReagentOne-step PrimeScript RT-PCR kit for real-time RT-PCR
ReagentOne Step TB Green™ PrimeScript™ RT-PCR Kit II Takara Bio Inc.Catalog #RR086A
Before start
have ice ready
wipe surfaces and pipettes with RNAse zap
prepare 70% ethanol
Cell harvesting and RNA extraction
Cell harvesting and RNA extraction
Aspirate medium from 6 well plates, wash once with PBS

Add Amount500 µL lysis buffer containig freshly added beta-mercaptoethanol to each well (lysis buffer provided in RNAeasy kit)

Note
Beta-mercaptoethanol should be added freshly every time performing the experiment

(OPTIONAL) Wrap plates in parafilm, store atTemperature-80 °C until extraction

(Defrost plates and) Scrap with 1000 ul pipette tip and pipett up and down the well, transfer into low bind nuclease-free tube
Extract according to manufacturer's protocol using Qiagen RNAeasy kit (including DNAse treatment step)
1h
Storage
Storage
Measure RNA concentration using Nanodrop and store atTemperature-80 °C or continue with qPCR

qPCR
qPCR
Dilute samples to have equal concentrations of 2500 ng of each sample.
Use Amount2 µL of this diluted RNA directly in 10 ul reaction to achieve 50nM per well of 384 well reaction plate

Mix contents of One Step TB Green™ PrimeScript™ RT-PCR Kit II (in case running a whole 384well plate)

Pre Master: Mix contents of entire kit:
  • Amount2500 µL TB Green buffer
  • Amount200 µL RT/Polymerage enzyme
  • Amount100 µL ROX
  • Amount800 µL H2O
  • Amount3600 µL total

Adjust accordingly to the total number of required reactions

1x Mastermix:
  1x in micro-liters
ENZYME 0.4
TB green Buffer 4  5
Primer 30uM F/R mix 0.8
RNA 2
ROX 0.4
H2O 1.8

Final master mix:

Per reaction/ per well of 384well plate = Amount10 µL
Multiply by amount of samples and add ~20% dead volume.