Apr 16, 2023
RNA extraction from hairy roots of common bean (Phaseolus vulgaris L.) and cDNA synthesis
- 1Biotechnology Institute, National Autonomous University of Mexico;
- 2Instituto de Biotecnología, Universidad Nacional Autónoma de México
Protocol Citation: Ronal Pacheco, Noreide Nava 2023. RNA extraction from hairy roots of common bean (Phaseolus vulgaris L.) and cDNA synthesis. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5jq24l1b/v1
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: April 10, 2023
Last Modified: April 16, 2023
Protocol Integer ID: 80277
Abstract
Extracting RNA for subsequent quantification of transcript levels by RT-qPCR requires high purity and concentration. When the amount of tissue is not abundant, as is the case with hairy roots, the concentration of RNA is frequently low. Here we present an optimized protocol for TRIzol-mediated RNA extraction from hairy roots of common bean. This protocol is based on the manufacturer's instructions TRIzol ReagentThermo Fisher ScientificCatalog #15596026
Materials
QIAzol Lysis ReagentCatalog #79306
Protocol materials
TRIzol ReagentThermo Fisher ScientificCatalog #15596026
Abstract, Step 2
QIAzol Lysis ReagentCatalog #79306
Materials
NanoDrop™ 2000c SpectrophotometerThermo Fisher ScientificCatalog #ND-2000C
Step 22
DNase I recombinant RNase-freeMerck MilliporeSigma (Sigma-Aldrich)Catalog #04716728001
Step 24
RevertAid Reverse Transcriptase (200 U/µL)Thermo FisherCatalog #EP0442
Step 28
Extraction of total RNA
Extraction of total RNA
Macerate root tissue using liquid nitrogen.
Load 100 mg of macerated tissue into a 1.5 mL Eppendorf tube and add 1 mL of TRIzol ReagentThermo Fisher ScientificCatalog #15596026 .
Note
If the amount of root tissue is less than 100 mg, add the equivalent amount of Trizol.
Mix by vortexing00:00:15 and incubate for 00:05:00 at room temperature.
5m 15s
Add200 µL chloroform:isoamyl alcohol 24:1 , mix by vortexing 00:00:15 and incubate for 00:03:00 at room temperature.
3m 15s
Centrifuge 11800 rpm, 4°C, 00:15:00
15m
Transfer the aqueous phase to a new 1.5 mL Eppendorf tube.
Add 500 µL of isopropanol, mix by immersion, and incubate Overnight -20 °C .
Note
The original protocol indicates incubating for 10 min at room temperature; however, we have had a low RNA concentration using these conditions. We strongly recommend incubating for at least 6 h at -20°C to get a higher concentration of RNA.
10m
Centrifuge 11800 rpm, 4°C, 00:10:00
10m
Transfer the aqueous phase to a new 1.5 mL Eppendorf tube.
Add 500 µL of 4 M LiCl and rise the pellet, do not resuspend, vortex slowly.
Note
LiCl increased the RNA concentration; thereby, this is an important step to reaching a high concentration of RNA.
Centrifuge 5900 rpm, 4°C 00:20:00
20m
Discard the LiCl phase.
Note
LiCl is difficult to remove; so, try to remove all remanents using a micropipette or syringe.
Add 500 µL of tris-EDTA buffer 8 . Resuspend RNA by vortexing.
Add 500 µL chloroform:isoamyl alcohol 24:1 v/v and mix by vortexing.
Centrifuge 5900 rpm, 4°C, 00:10:00
10m
Transfer the aqueous phase to a new 1.5 mL Eppendorf tube.
Add 500 µL of isopropanol and 66 µL of 3 M sodium acetate 5.2 Mix by immersion and incubate Overnight -20 °C
Note
The original protocol does not include an overnight incubation step, but we strongly recommend incubating for at least 6 h at -20°C to get a higher concentration of RNA.
Centrifuge 11800 rpm, 4°C, 00:10:00
10m
Discard the aqueous phase and vacuum or air dry the RNA pellet.
Resuspend RNA pellets using nuclease-free water. Preferably, use DEPC-treated water.
Preparation of RNA samples for cDNA synthesis
Preparation of RNA samples for cDNA synthesis
35m
35m
Check the integrity of RNA in a 1% agarose gel treated with bleach.
CITATION
Prepare a dilution (1/10) of each RNA sample and quantify the concentration using aNanoDrop™ 2000c SpectrophotometerThermo Fisher ScientificCatalog #ND-2000C or an equivalent instrument.
Prepare one aliquot 10 µL of each RNA sample at 10 ng/μl
Add 1 µL of DNase I recombinant RNase-freeMerck MilliporeSigma (Sigma-Aldrich)Catalog #04716728001
and 1 µL of the corresponding buffer (10X) to each RNA sample.
Note
To reduce pipetting errors, prepare a mix of the DNase and the incubation buffer 1:1 v/v (total volume according to the number of samples) and add 2 µL of this mix to each RNA sample.
Incubate samples00:30:00 37 °C
Note
After this step, a qPCR assay should be performed to check if traces of genomic DNA are remaining. According to our experience, DNase I recombinant RNase-freeMerck MilliporeSigma (Sigma-Aldrich)Catalog #04716728001 efficiently remove all traces of genomic DNA by performing this step.
30m
Add 1 µL of 10 millimolar (mM) oligo (dT) to each sample and incubate00:05:00 70 °C , then immediately incubate On ice 3 m .
5m
Synthesis of cDNA
Synthesis of cDNA
1h 40m
1h 40m
Prepare a mix containing 1 µL ofRevertAid Reverse Transcriptase (200 U/µL)Thermo FisherCatalog #EP0442 , 4 µL of the corresponding buffer (5X), and 2 µL dNTP mix . Add 7 µL of the mix to each RNA sample.
Incubate 01:30:00 at42 °C and 00:10:00 70 °C .
1h 40m
Store cDNA samples at -20 °C .
Protocol references
Bleach gel: A simple agarose gel for
analyzing RNA quality