Jan 21, 2025
Version 1
RNA Extraction and QC V.1
This protocol is a draft, published without a DOI.
- Raphael Joshua De Guzman1
- 1St. Luke's Medical Center
Protocol Citation: Raphael Joshua De Guzman 2025. RNA Extraction and QC . protocols.io https://protocols.io/view/rna-extraction-and-qc-dvnn65de
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: In development
We are still developing and optimizing this protocol
Created: December 19, 2024
Last Modified: January 21, 2025
Protocol Integer ID: 116142
Disclaimer
None
Abstract
Masters Thesis
microRNA extraction protocols
microRNA extraction protocols
Equipment Preparation
Autoclave Pipettor Tips, Motar and Pestle, Forceps, Razor blades, funnels
Prepare Weigh boat,
Decotaminate Lab bench and pipettors with RNAse away
Prechill mortar and pestle with dry ice or liquid nitrogen
Dry Bath, Vortex, Thermomixer
Prepare Samples (Benchtop)
Measure or estimate the weight of the sample. 0.5–250 mg of tissue needed for processing
Place 10 volumes of Lysis/Binding Buffer per tissue mass into a tube on ice.
We suggest using a weigh boat because it is easier to transfer frozen powdered tissue to a weigh boat than to a tube of Lysis/Binding buffer.
Note: 1 mL Lysis/Binding Buffer per 0.1 g of tissue
Grind frozen tissue to a powder with liquid nitrogen in a prechilled mortar and pestle sitting in a bed of dry ice.
Using a prechilled metal spatula, scrape the powdered tissue into the Lysis/Binding Buffer, and mix rapidly.
Transfer the mixture to a vessel for homogenization and process the mixture to homogeneity, i.e. until all visible clumps are dispersed. If available, use a motorized rotor-stator homogenizer (e.g. Polytron).
Perform Organic Extraction (To be performed in BSC II)
Add 1/10 volume of miRNA Homogenate Additive to the cell or tissue lysate (or homogenate), and mix well by vortexing or inverting the tube several times.
For example, if the lysate volume is 300 μL, add 30 μL miRNA Homogenate Additive.
Leave the mixture on ice for 10 min.
For this step, perform under a hood and wear N95
Add a volume of Acid-Phenol:Chloroform that is equal to the lysate volume before addition of the miRNA Homogenate Additive.
For example, if the original lysate volume was 300 μL, add 300 μL Acid-Phenol:Chloroform.
Be sure to withdraw from the bottom phase in the bottle of Acid-Phenol:Chloroform, because the upper phase consists of an aqueous buffer.
Vortex for 30–60 sec to mix.
Centrifuge for 5 min at maximum speed (10,000 x g) at room temperature to separate the aqueous and organic phases. After centrifugation, the interphase should be compact; if it is not, repeat the centrifugation.
Carefully remove the aqueous (upper) phase without disturbing the lower phase, and transfer it to a fresh tube. Note the volume removed.
Prepare for Final RNA Isolation
Preheat Elution Solution or nuclease-free water to 95°C for use in eluting the RNA from the filter at the end of the procedure.
If the 100% ethanol you plan to use for this procedure is stored cold, warm it to room temperature before starting the Final RNA Isolation.
Enrichment procedure for small RNAs
Add 1/3 volume of 100% ethanol to the aqueous phase recovered from the organic extraction (e.g. add 100 μL 100% ethanol to 300 μL aqueous phase). Mix thoroughly by vortexing or inverting the tube several times.
For each sample, place a Filter Cartridge into one of the Collection Tubes supplied.
Pipet the lysate/ethanol mixture (from the previous step) onto the Filter Cartridge. Up to 700 μL can be applied to a Filter Cartridge at a time. For sample volumes greater than 700 μL, apply the mixture in
successive applications to the same filter.
Centrifuge for ~15 sec to pass the mixture through the filter. Centrifuge at RCF 10,000 x g (typically 10,000 rpm). Spinning harder than this may damage the filters.
Alternatively, vacuum pressure can be used to pull samples through the filter.
Collect the filtrate. If the lysate/ethanol mixture is >700 μL, transfer the flow-through to a fresh tube, and repeat until all of the lysate/ethanol mixture is through the filter. Pool the collected filtrates if multiple passes were done, and measure the total volume of the filtrate.
Add 2/3 volume room temperature 100% ethanol to filtrate (i.e. flow-through). For example, if 400 μL of filtrate is recovered, add 266 μL 100% ethanol. Mix thoroughly.
For each sample, place a Filter Cartridge into one of the Collection Tubes supplied.
Pipet the filtrate/ethanol mixture (from the previous step) onto a second Filter Cartridge. Up to 700 μL can be applied to a Filter Cartridge at a time. For sample volumes greater than 700 μL, apply the mixture in successive applications to the same filter.
Centrifuge for ~15 sec to pass the mixture through the filter. Centrifuge at RCF 10,000 x g (typically 10,000 rpm). Spinning harder than this may damage the filters.
Discard the flow-through, and repeat until all of the filtrate/ethanol mixture is through the filter. Reuse the Collection Tube for the washing steps.
Apply 700 μL miRNA Wash Solution 1 (working solution mixed with ethanol) to the Filter Cartridge and centrifuge for ~5–10 sec. Discard the flow-through from the Collection Tube, and replace the Filter Cartridge into the same Collection Tube.
Apply 500 μL Wash Solution 2/3 (working solution mixed with ethanol) and draw it through the Filter Cartridge as in the previous step.
Repeat with a second 500 μL aliquot of Wash Solution 2/3.
After discarding the flow-through from the last wash, replace the Filter Cartridge in the same Collection Tube and spin the assembly for 1 min to remove residual fluid from the filter.
Transfer the Filter Cartridge into a fresh Collection Tube (provided with the kit). Apply 100 μL of pre-heated (95°C) Elution Solution or nuclease-free water to the center of the filter, and close the cap. Spin for
~20–30 sec at maximum speed to recover the RNA.
Collect the eluate (which contains the RNA) and store it at –20°C or colder.
Analyze RNA after purification via Spectrophotometer
Dilute the sample 1:50 to 1:500 in water to bring the concentration into
the linear range of the spectrophotometer
Blank the spectrophotometer with water.
Read the absorbance at 260 nm and 280 nm The purity of the RNA can be assessed from the ratio of A260/A280. For highly pure RNA a ratio of 1.8–2.1 is expected.
Note: 100ng - 1ug of total RNA
Analyze miRNA quantitation via Qubit miRNA
Prepare the Qubit‱ working solution by diluting the Qubit‱ microRNA reagent 1:200 in the Qubit‱
microRNA buffer. Use a clean plastic tube each time you prepare a new Qubit‱ working solution.
Add the Qubit‱ microRNA working solution to each tube such that the final volume is 200 µL.
microRNA working solution to each tube such that the final volume is 200 µL
Vigorously vortex for 3–5 seconds.
Allow all tubes to incubate at room temperature for 2 minutes, then proceed to read standards and samples (next section).
On the Home screen, touch RNA, then select microRNA as the assay type. Touch Read standards to proceed.
Insert the tube containing Standard #1 into the sample chamber, close the lid, then touch Read standard. When the reading is complete (~3 seconds), remove Standard #1.
Insert the tube containing Standard #2 into the sample chamber, close the lid, then touch Read standard. When the reading is complete, remove Standard #2.
Touch Run samples.
On the assay screen, select the Sample volume and units. Touch the + or – buttons on the wheel, or anywhere on the wheel itself, to select the sample volume added to the assay tube
(1–20 μL). From the Unit dropdown menu, select the units for the output sample concentration.
Insert a sample tube into the sample chamber, close the lid, then touch Read tube. When the reading is complete (~3 seconds), remove the sample tube. The top value (in large font) is the concentration of the original sample and the bottom value is the dilution concentration.
Repeat until all samples have been read.
Note: 10-100ng is recommended for NEBNext kit
Library Preparation Protocols
Library Preparation Protocols
Ligate 3' Adaptor
Mix the following components in a sterile nuclease-free PCR tube. It is ok to premix the reagents. Use immediately.
Incubate in a preheated thermal cycler for 2 minutes at 70°C. Transfer tube to ice.
Add and mix the following components. It is ok to premix the reagents. Use immediately
Incubate for 1 hour at 25°C in a thermal cycler.
Hybridize the Reverse Transcription Primer
Add and mix the following components to the ligation mixture from Step 1.4 and mix well. It is ok to premix the reagents.
Place in a thermocycler with heated lid set to > 85°C and run the following program:
5 minutes at 75°C
15 minutes at 37°C
15 minutes at 25°C
Hold at 4°C
Ligate the 5´ SR Adaptor
With 5 minutes remaining, resuspend the (yellow) 5´ SR adaptor in 120 μl of nuclease free water.
Note: For total RNA inputs closer to 100 ng, additionally dilute the (yellow) 5´ SR Adaptor for Illumina 1:2 in nuclease free water. For total RNA inputs closer to 1 µg do not dilute the adaptor further.
Aliquot the (yellow) 5´ SR Adaptor into a separate, nuclease-free 200 µl PCR tube, for the number of samples in the experiment plus an excess of 10%
Incubate the adaptor in the thermal cycler at 70°C for 2 minutes and then immediately place the tube on ice. Keep the tube on ice and use the denatured adaptor within 30 minutes of denaturation.
Add and mix the following components to the ligation mixture from Step 2.2 and mix well. Do not premix reagents
Incubate for 1 hour at 25°C in a thermal cycler.
Perform Reverse Transcription
Mix the following components in a sterile, nuclease-free tube. It is ok to premix the reagents. Use immediately.
Incubate for 60 minutes at 50°C.
Immediately proceed to PCR amplifcation.
Safe Stopping Point: If you do not plan to proceed immediately to PCR amplifcation, then heat inactivate the RT reaction at 70°C for 15 minutes. Samples can be safely stored at –15°C to –25°C.
Perform PCR Amplification
Add and mix the following components to the RT reaction mix from Step 11 and mix well:
*Note: The NEBNext Multiplex Small RNA Library Prep Set for Illumina Set 1 (E7300) contains 1–12 PCR primers, each with a different index. For each reaction, only one of the 12 PCR primer; the NEBNext Multiplex Small RNA Library Prep Set for Illumina Set 2 (E7580) contains 13-24 PCR primers;
Perform PCR Amplification
This protocol was optimized using 1 µg of total RNA from human brain and 12 PCR cycles. If less small RNA, 15 cycles; if more small RNA, less than 12 cycles
Safe Stopping Point: It is safe to store the library at -20°C after PCR. Avoid leaving the sample at 4°C overnight if possible.
Quality Control
Quality Control
Purify the PCR amplified cDNA construct (100 µl) using a Monarch PCR & DNA Kit
IMPORTANT: Use the 7:1 ratio of binding buffer:sample.Discard the flow through after each centrifugation step.
Elute amplified DNA in 27.5 µl Nuclease-free Water.
Safe Stopping Point: It is safe to store the library at -20°C.
Load 1 µl of the purified PCR reaction on the Bioanalyzer using a DNA 1000 chip according to the manufacturer's instructions
(Figure)
The 143 and 153 bp bands correspond to miRNAs and piRNAs, respectively. miRNA peak should be ~ 143-146 bp.
Mix the purified PCR product (25 µl) with 5 µl of Gel Loading Dye, Blue (6X).
Note: Vortex the Gel Loading Dye, Blue throughly to mix well before using
Load 5 µl of Quick-Load pBR322 DNA-MspI Digest in one well on the 6% PAGE 10-well gel.
Load two wells with 15 µl each of mixed amplified cDNA construct and loading dye on the 6% PAGE 10-well gel.
Run the gel for 1 hour at 120 V or until the blue dye reaches the bottom of the gel. Do not let the blue dye exit the gel.
Remove the gel from the apparatus and stain the gel with SYBR Gold nucleic acid gel stain in a clean container for 2–3 minutes and view the gel on a UV transiluminator (Figure 2).
The 140 and 150 bp bands correspond to miRNAs (21 nt) and piRNAs (30 nt), respectively.
For miRNAs, isolate the bands corresponding to ~140 bp
Place the two gel slices from the same sample in one 1.5 ml tube and crush the gel slices with the RNase-free Disposable Pellet Pestles and then soak in 250 µl DNA Gel Elution buffer (1X).
Rotate end-to-end for at least 2 hours at room temperature.
Transfer the eluate and the gel debris to the top of a gel filtration column
Centrifuge the filter for 2 min at > 13,200 rpm.
Recover eluate and add 1 µl Linear Acrylamide, 25 µl 3M sodium acetate, pH 5.5 and 750 µl of 100% ethanol.
Vortex well.
Precipitate in a dry ice/methanol bath or at –80°C for at least 30 minutes.
Spin in a microcentrifuge @ > 14,000 x g for 30 minutes at 4°C.
Remove the supernatant taking care not to disturb the pellet.
Wash the pellet with 80% ethanol by vortexing vigorously.
Spin in a microcentrifuge @ > 14,000 x g for 30 minutes at 4°C.
Air dry pellet for up to 10 minutes at room temperature to remove residual ethanol.
Resuspend pellet in 12 µl TE Buffer.
Load 1 µl of the size selected purified library on a 2100 Bioanalyzer using a DNA 1000 or High Sensitivity DNA chip according to the manufacturer's instructions
Check the size, purity, and concentration of the sample.
Electropherogram trace of the gel size selected purified library from human brain total RNA.