Protocols_RNA Quantification_ Quality Control Protocol - BackmanLab/BackmanLab.github.io GitHub Wiki

Procedure: RNA Quantification/ Quality Control

SOP Prepared by: Emily Pujadas Version: 1.0

Scope

The purpose of this SOP is to describe how to ensure that only RNA of high purity and integrity is used in RT-qPCR experiments after total RNA extraction.

Description

This protocol aims to describe the procedure of RNA quantification and quality control. Ensuring that only RNA of high purity (no contaminants) and high integrity (not degraded) is used is one of the most critical points in the RT-qPCR experimental workflow. Impurities in the RNA sample may lead to inhibition of the RT and PCR can lead to varying and incorrect quantification results. [An OD (260/280) of 1.8-2.0 indicates good RNA quality that is devoid of protein and phenol contamination.]{.ul} RNA integrity information can be obtained with a bioanalyzer. The analyzer measures the sizes of the rRNA bands and determines an RNA Integrity Number (RIN) to standardize between RNA samples. [While the values of the 28S/18S ratio can vary between different cell types, the Bioanalyzer system provides a RIN (RNA Integrity Number) value, an objective metric of total RNA quality ranging from 10 (highly intact RNA) to 1 (completely degraded RNA)]{.ul}

Personal Protective Equipment

gloves

Potential Hazards + Safety Precautions

Nitrile gloves should be used at all times.
RNaseZap RNase Decontamination solution should be used to ensure an RNase-free environment. This can be used for cleaning work surfaces, pipettors, and equipment that must be RNase free
Kit components contain DMSO. Because the dye binds to nucleic acids, it should be treated as a potential mutagen and used with appropriate care. Wear hand and eye protection and follow good laboratory practices when preparing and handling reagents and samples.

Materials and Reagents

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Equipment

= RNA Quantification/ Quality Control (Cont.) =

Nanodrop Spectrophotometer (Thermo Fisher Scientific, Cat# ND2000

-- Use in rPPC, must request training in advance)
Bioanalyzer 2100 (Agilent, Part# G2929BA -- Stored in Hi-C room)
RNA 6000 Nano Kit (Agilent, Cat# 5067-1511 -- Chips stored in Hi-C

room, Reagents stored at 4 C in Tissue Culture Room)
Heating block for ladder/ sample denaturation
IKA vortex mixer (Model MS3)
Chip priming station (Agilent, Part# 5065-4401, Stored in Hi-C

room)
16-pin bayonet electrode cartridge (Agilent, Part# 5065-4413,

Stored in Hi-C room)

Materials

RNaseZap RNase Decontamination Solution (Invitrogen, Cat# AM9780 --

Stored at RT in Hi-C room)
Pipettes and tips
1.5 ml microcentrifuge tubes (RNase-free)

Reagents and Solutions

RNase-free water

= RNA Quantification/ Quality Control (Cont.) =

Procedure

Protocol for Measuring RNA Quantity:

Bring RNA samples as well as the water used to elute them on ice to the spectrophotometer
Wash the sample reader with molecular grade water and dry with a KimWipe
Following the software's instructions, load 2 µL of elution water (blank) and initialize the system
Change the computer's setting to RNA and click the blank button
Load 2 µL of sample and click the measure button
After the read is complete, record the A260/A280 and A260/A230 ratios as well as the amount of RNA recovered (in ng/µL)

Very pure RNA will have an A260/A280 ratio of ~2.1. Anything higher than 1.8 is considered to be of acceptable purity, and a ratio of <1.8 indicates potential DNA or protein contamination.
The A260/A230 ratio should also be above 2.0.

Wipe the sample reader with a clean, dry KimWipe between samples and repeat steps 5-6.

Things to do Before Measuring RNA Integrity:

Before beginning the chip preparation protocol, ensure that the chip priming station and the bioanalyzer are set up and ready to use. You have to:

replace the syringe at the chip priming station with each new DNA kit
adjust the base plate of the chip priming station [(set to position C)]{.ul}
adjust the syringe clip at the chip priming station
adjust the bioanalyzer's chip selector
set up the vortex mixer
finally, make sure that you start the software before you load the chip.

Start the 2100 Expert Software, ensure that the lid is closed, chip inserted, RNA or demo assay selected
Prepare the RNA Ladder after arrival

Pipette the ladder in RNase-free vial
Heat denature it for 2 min at 70 C
Immediately cool down the vial on ice
Prepare aliquots in RNase-free vials with the required amount for a typical daily use
Store aliquots at -70 C
Before use, thaw ladder aliquots and keep them on ice (avoid extensive warming upon thawing process)

Decontaminate the electrodes

Slowly fill one of the wells of an electrode cleaner with 350 ul RNaseZAP
Open the lid and place electrode cleaner in the Agilent 2100 bioanalyzer.
Close the lid and leave it closed for about 1 min
Open the lid and remove the electrode cleaner. Label the electrode cleaner and keep it for future use. You can reuse the electrode cleaner for all 25 chips in the kit.
Slowly fill one of the wells of another electrode cleaner with 250 ul of RNase-free water.
Place electrode cleaner in the Agilent 2100 bioanalyzer.
Close the lid and leave it closed for about 10 sec
Open the lid and remove the electrode cleaner. Label it and keep it for future use.
Wait another 10 sec for the water on the electrodes to evaporate before closing the lid.

NOTE 1: Allow all reagents and samples to equilibrate to room temperature for 30 min before use.

NOTE 2: Protect dye and dye mixtures from light. Remove light covers only when pipetting. The dye decomposes when exposed to light and this reduces the signal intensity.

NOTE 3: Always insert the pipette tip to the bottom of the well when dispensing the liquid. Placing the pipette at the edge of the well may lead to poor results.

NOTE 4: It is recommended to head denature all RNA samples and RNA ladder before use.

NOTE 5: Always vortex the dye concentrate for 10 sec before preparing the gel-dye mix.

NOTE 6: Use a new syringe and electrode cleaners with each new kit.

NOTE 7: Use loaded chips within 5 min because reagents might evaporate, leading to poor results.

Protocol for Measuring RNA Integrity:

Prepare the gel

Allow all reagents to equilibrate to room temperature for 30 minutes before use.
Place 550 μl of Agilent RNA 6000 Nano gel matrix (red) into the top receptacle of a spin filter.
Place the spin filter in a microcentrifuge and spin for 10 minutes at 1500 g ± 20 % (for Eppendorf microcentrifuge, this corresponds to 4000 rpm).
Aliquot 65 μl filtered gel into 0.5 ml RNase-free microfuge tubes that are included in the kit. Store the aliquots at 4 °C and use them within one month of preparation.

Prepare the gel-dye mix

Allow all reagents to equilibrate to room temperature for 30 minutes before use. Protect the dye concentrate from light while bringing it to room temperature.
Vortex RNA 6000 Nano dye concentrate (blue) for 10 seconds and spin down.
Add1μlofRNA6000Nanodye concentrate (blue) to a 65 μl aliquot of filtered gel
Cap the tube, vortex thoroughly and visually inspect proper mixing of gel and dye. Store the dye concentrate at 4 °C in the dark again.
Spin tube for 10 minutes at room temperature at 13000 g (for Eppendorf microcentrifuge, this corresponds to 14000 rpm). Use prepared gel-dye mix within one day.

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Load the gel-dye mix

Allow the gel-dye mix to equilibrate to room temperature for 30 minutes before use and protect the gel-dye mix from light during this time.
Take a new RNA Nano chip out of its sealed bag.
Place the chip on the chip priming station.
Pipette 9.0 μl of the gel-dye mix at the bottom of the well marked G and dispense the gel-dye mix.

{width="1.1176476377952755in" height="1.1176476377952755in"}

Set the timer to 30 seconds, make sure that the plunger is positioned at 1 ml and then close the chip priming station. The lock of the latch will click when the Priming Station is closed correctly.
Press the plunger of the syringe down until it is held by the clip.
Wait for exactly 30 seconds and then release the plunger with the clip release mechanism.
Visually inspect that the plunger moves back at least to the 0.3 ml mark.

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Wait for 5 seconds, then slowly pull back the plunger to the 1 ml position.
Open the chip priming station.
Pipette 9.0 μl of the gel- dye mix in each of the wells marked.

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NOTE 8: Discard the remaining vial with gel-dye mix.

Load the RNA 6000 Nano Marker

Pipette 5 μl of the RNA 6000 Nano marker (green ) into the well marked with the ladder symbol and each of the 12 sample wells.

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Note 9: Do not leave any wells empty or the chip will not run properly. Unused wells must be filled with 5 μl of the RNA 6000 Nano marker (green ) plus 1 μl of the buffer in which the samples are diluted.

Note 10: Always use RNase-free microfuge tubes, pipette tips and water.

Load the ladder and samples

Before use, thaw ladder aliquots and keep them on ice (avoid extensive warming upon thawing process)
To minimize secondary structure, heat denature (70 °C, 2 minutes) the samples before loading on the chip.
Pipette 1 μl of the RNA ladder into the well marked with the ladder symbol.

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Pipette 1 μl of each sample into each of the 12 sample wells.

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Set the timer to 60 sec
Place the chip horizontally in the adapter of the IKA vortex mixer and make sure not to damage the buldge that fixes the chip during vortexing. If there is liquid spill at the top of the chip, carefully remove it with a tissue
Vortex for 60 seconds at 2400 rpm.
Refer to the next topic on how to insert the chip in the Agilent 2100 bioanalyzer. Make sure that the run is started within 5 minutes.

NOTE 11: Depending on the RNA isolation protocol, varying results can be expected. Known dependencies include: salt content, cell fixation method and tissue stain. Best results are achieved for samples which are dissolved in deionized and RNase-free water. Avoid genomic DNA contamination by including DNase treatment in the preparation protocol.

Insert chip in the Agilent 2100 Bioanalyzer

Open the lid of the Agilent 2100 bioanalyzer.
Check that the electrode cartridge is inserted properly and the chip selector is in position
Place the chip carefully into the receptacle. The chip fits only one way.
Carefully close the lid. The electrodes in the cartridge fit into the wells of the chip.
The 2100 expert software screen shows that you have inserted a chip and closed the lid by displaying the chip icon at the top left of the Instrument context.

Start the chip run

In the Instrument context, select the appropriate assay from the Assay menu.
Accept the current File Prefix or modify it. Data will be saved automatically to a file with a name using the prefix you have just entered. At this time you can also customize the file storage location and the number of samples that will be analyzed.
Click the Start button in the upper right of the window to start the chip run. The incoming raw signals are displayed in the Instrument context.
To enter sample information like sample names and comments, select the Data File link that is highlighted in blue or go to the Assay context and select the Chip Summary tab. Complete the sample name table.
To review the raw signal trace, return to the Instrument context.
After the chip run is finished, remove the chip from the receptacle of the bioanalyzer and dispose it according to good laboratory practices

NOTE 12: Leaving the chip for a period longer than 1 hour (e.g. overnight) in the bioanalyzer may cause contamination of the electrodes. Immediately remove the chip after a run.

Clean up after the RNA 6000 Nano Chip run

Slowly fill one of the wells of the electrode cleaner with 350 μl RNase- free water.
Open the lid and place the electrode cleaner in the Agilent 2100 bioanalyzer.
Close the lid and leave it closed for about 10 seconds.
Open the lid and remove the electrode cleaner.
Wait another 10 seconds to allow the water on the electrodes to evaporate before closing the lid.

NOTE 13: After 5 chip runs, empty and refill the electrode cleaner. After 25 chi9p runs, replace the used electrode cleaner by a new one.

Checking RNA Integrity Results:

Check RNA 6000 Nano Ladder Well Results

To check the results of your run, select the Gel or Electropherogram tab in the Data context. The electropherogram of the ladder well window should resemble those shown below.

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Major features of a successful ladder run are:
- 1 marker peak
- 6 RNA peaks (2100 expert software calls for 5 first ladder peaks only)
- All 7 peaks are well resolved
- Correct peak size assignment in the electropherogram

{width="3.254695975503062in" height="1.9130708661417324in"}

Check RNA 6000 Nano Sample Well Results

To review the results of a specific sample, select the sample name in the tree view and highlight the Results sub- tab. The electropherogram of the sample well window for total RNA (eukaryotic) should resemble the one shown here.
Major features of a successful total RNA run are:
- 1 marker peak
- 2 ribosomal peaks (with successful sample preparation)

{width="3.601641513560805in" height="2.1339457567804025in"}

By selecting the Results sub- tab, values for the calculated RNA concentration, the ribosomal ratio and the RNA Integrity Number (RIN), implemented with 2100 expert software version B.02.02 or higher, are displayed.
The electropherogram of the sample well window for mRNA should resemble the one shown here.
Major features of a successful mRNA run are:
- 1 marker peak
- Broad hump (with successful sample preparation)
- Contamination with ribosomal RNA shown as 2 overlaid peaks (if present)

{width="3.5336679790026246in" height="2.082352362204724in"}

Disposals | Spills | Incidents

Waste (see waste segregations and collection SOP for complete information)

All waste containers should be closed once ¾ 's and placed into the appropriate location for pick up full.
All waste containers should be properly labelled with the following information:
- PI's name
- User | Responsible person
- Content
- Waste class (e.g. chemical, biohazard, radioactive, etc.)
= RNA Quantification/ Quality Control (Cont.) =
- Waste type (e.g. sharps, plastic, glass, metal, etc.)
- Waste "status" (e.g. liquid, solid, etc.)