Feb 12, 2025
Extraction of High-Quality RNA from Auxenochlorella protothecoides (UTEX 250)
- 1Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA
- Merchant Lab UC Berkeley
Protocol Citation: Dimitrios J. Camacho, Sabeeha S. Merchant 2025. Extraction of High-Quality RNA from Auxenochlorella protothecoides (UTEX 250). protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr8yk3lmk/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: August 31, 2024
Last Modified: February 12, 2025
Protocol Integer ID: 106782
Keywords: RNA, RNA extraction, bioanalyzer, RNA quality, Transcriptomics, Auxenochlorella, algae, Qubit, RNA Broad Range Assay, RNA 6000 Pico Kit, ZymoBIOMICS RNA Miniprep Kit
Funders Acknowledgements:
National Institutes of Health (NIH): Nutritional Copper Signaling and Homeostasis Grant
Grant ID: GM 042143
National Institutes of Health (NIH): Molecular Basis of Cell Function T32 Training Grant
Grant ID: 5T32GM007232-44
US Department of Energy (DOE), Office of Biological and Environmental Research (BER): Systems Engineering of Auxenochlorella protothecoides: from Photosynthesis to Biofuels and Bioproducts Grant
Grant ID: DE-SC0023027
University of California, Berkeley, Chancellor’s Fellowship
Grant ID: N/A
Disclaimer
This protocol uses the following kits and is an adaptation of the associated manuals.
ZymoBIOMICS RNA Miniprep Kit: https://files.zymoresearch.com/protocols/_r2001_zymobiomics_rna_miniprep_kit.pdf
Qubit RNA BR Assay Kit: https://assets.thermofisher.com/TFS-Assets/LSG/manuals/Qubit_RNA_BR_Assay_UG.pdf
Agilent RNA 6000 Pico Kit: https://www.agilent.com/cs/library/usermanuals/public/G2938-90046_RNA600Pico_KG_EN.pdf
Abstract
This protocol describes a reliable method for extracting high quality total RNA from Auxenochlorella protothecoides (UTEX 250). It covers the preparation of RNase-free clean materials, RNA extraction, accurate quantification, and quality assessment. This procedure utilizes the ZymoBIOMICS RNA Miniprep Kit for total RNA extraction, the Thermo Fisher Qubit system for accurate quantification of RNA, and the Agilent Bioanalyzer for evaluation of RNA quality. The protocol consistently produces high quality total RNA, suitable for preparing libraries used in long-read sequencing (PacBio Iso-Seq, Oxford Nanopore Technologies Direct RNA-Seq, etc.) and short-read sequencing (Illumina RNA-Seq).
Guidelines
RNA is highly susceptible to degradation by RNases. RNases are robust, stable, and abundant. Assume they are present on all surfaces and items. They are extremely difficult to inactivate. Autoclave sterilization and RNase inhibitors do not completely inactivate them. RNases can be eliminated either by 0.3 M NaOH or by baking RNase exposed materials (no plastics) in an oven overnight (6 h minimum) at 250 °C
Apply the following practices to avoid RNA degradation:
Avoid working in areas that have been used for DNA extraction. DNA extraction protocols use RNase.
Create an RNA work zone (see the "Before Start" section).
Use nuclease free sterile water which can be sourced from a Milli-Q water purification system.
Wrap metal tools and glassware in aluminum foil and bake overnight (6 h minimum) at 235 °C
Use pipettors dedicated for RNA work.
Clean pipettors with an RNase inhibitor or killer cocktail and rinse with nuclease free water.
Use sterile nuclease-free pipette filter tips.
Change gloves frequently.
If touching contaminated surfaces, use an RNAse free Kimwipe between your gloves and the surface.
Never reach into a bag of tubes. Shake and pour tubes out onto a clean surface. Massage the outside of bags to dispense individual tubes or columns. Do not touch surfaces that will come into contact with the sample such as pipette tips and the insides of tubes and caps.
Use fresh Auxenochlorella cells that have not been previously frozen.
Store RNA aliquots at -80 °C and avoid repeated freezing and thawing.
Some RNA sequencing facilities do not accept RNA quantified solely by A260 optical absorbance (e.g. NanoDrop). They often require precise quantification by a Qubit and quality assessment by a Bioanalyzer or TapeStation. In some cases, these services may be provided by the sequencing facility itself. The Agilent Bioanalyzer has been discontinued and replaced with the Agilent TapeStation.
Materials
Equipment:
Microscope and hemocytometer
Beckman Coulter Avanti JXN-26 centrifuge
Fixed angle centrifuge rotor JA-14.50 with 15 mL adapters
Liquid waste container
Mini-Beadbeater-16, Biospec Products, (Cat. No. 607)
4°C cold room
RAININ P1000 pipette and low adhesion tips
RAININ P200 pipette and low adhesion tips
RAININ P20 pipette and low adhesion tips
RAININ P2 pipette and low adhesion tips
Timer with seconds displayed
Flexible plastic spatulas
Centrifuge Eppendorf, model 5810 R (with A-4-81 15/50 mL swinging bucket rotor)
Centrifuge Eppendorf, model: 5424R (with FA-45-24-11 2 mL fixed angle rotor)
Vortex mixer, Corning, REF: 6775
Computer with 2100 Expert Software installed
IKA MS3 vortex mixer
Chip priming station (reorder-no 5065-4401)
Qubit 4 fluorometer, Thermofisher Scientific, REF: Q33226
Nanodrop, Thermofisher Scientific, model: NanoDrop One / NanoDrop One C
T100 Thermal Cycler, BioRad
70 °C Heating Block
Kits:
ZymoBIOMICS RNA Miniprep Kit (Cat. No. R2001)
ThermoFisher Scientific RNA Quantification, broad range, 100 reactions (Cat. No. Q10210)
Agilent RNA 6000 Pico Kit (reorder-no 5067-1513)
Consumables:
0.3 M NaOH
100% ethanol
RNAse free H2O
100% isopropanol
RNAseZAP (Ambion, Inc., Cat. No. 9780)
15 mL conical tubes, Centrifuge, high performance, red screw cap, assembled, polypropylene, printed graduations, sterile, 25 tubes / bag, 20 bags/ case. (Globe Scientific inc., Cat. No. 6295), with a maximum rating of 17,000 ×g.
1.5 mL Eppendorf DNA LoBind microfuge tubes, (FIsher Scientific, Cat. No. 13-698-791).
Qubit Assay Tubes (Thermo Fisher Scientific, Cat. No. Q32856).
0.2 mL Flat PCR Tube 8-Cap Strips, optical, clear (BioRad, Cat.No. TCS0803)
Whatman Benchkote Surface Protectors "Elephant paper" 460 × 570 mm (100 pcs) (Cytiva, Cat. No. 2300-917)
Kimtech Science Kimwipes Delicate Task Wipes (16.4"×14.43") (Kimberly-Clark Professional, Cat. No. 34256)
Safety warnings
Read the SDS sheets for all kits and reagents before use.
NaOH
NaOH may cause skin irritation, eye damage, and corrode work surfaces. Rinse all exposed surfaces with nuclease free water immediately. Violent reactions may occur with NaOH and many other chemicals.
ZymoBIOMICS RNA Miniprep Kit
The zymoBIOMICS RNA Miniprep Kit contains the following hazardous reagents:
RNA Shield - May cause skin and eye irritation. Harmful if inhaled or ingested.
RNA Lysis Buffer - May cause severe burns and serious eye damage. Harmful if inhaled or ingested.
RNA Prep Buffer - Causes skin irritation and serious eye damage. Liquid and vapor is highly flammable. Harmful if inhaled or ingested.
RNA Wash Buffer - Becomes flammable after adding 96 mL of EtOH.
Agilent RNA 6000 Pico Kit and ThermoFisher Qubit RNA Broad Range Assay Kit
The Agilent RNA 6000 Pico Kit and ThermoFisher Qubit RNA Broad Range Assay Kit contain dyes that bind nucleic acids and should be treated as potential mutagens. The kits also contain DMSO, a flammable solvent that readily penetrates skin and can transport other dissolved chemicals into the body. The Agilent RNA 6000 Pico Kit also contains the following hazardous reagent:
RNA Pico Dye Concentrate - Flammable, causes eye irritation, and is hazardous to aquatic life.
Liquid Nitrogen
Liquid nitrogen may cause asphyxiation if the workspace is not adequately ventilated. Do not accompany liquid nitrogen dewars greater than 10 L in an elevator. Instead, send the dewar alone, secured, and place a clearly visible "Warning: Do Not Enter - Liquid Nitrogen in Transit" sign on the dewar. Direct contact with liquid nitrogen or vapors may cause frostbite. Wear a cryogen-compatible splash guard, apron, and insulated gloves to handle liquid nitrogen. Never use nitrile or latex gloves when handling liquid nitrogen, as they can trap the liquid and prevent it from safely evaporating off of skin. Never cap tubes tightly after flash-freezing, as trapped gas expansion during warming can cause explosions. Use vented caps or leave caps loosely fitted.
Do not touch the Bioanalyzer while it is running. Avoid vibrations near the machine.
Before start
Make sure that all metadata pertaining to the experiment has been properly recorded.
Prepare < 5 L of liquid nitrogen in a dewar and set aside a tube rack (for 1.5 mL tubes) that can be filled with liquid nitrogen.
Prepare wet ice.
RNA work zone
Prepare all necessary waste containers. This method does not use phenol or chloroform.
Choose a workspace that is free of dust and human traffic. Label the boundaries of this workspace "RNA work zone". Remind others in the lab that they should not touch this workspace unless they follow proper RNA handling procedures.
Wipe down all work surfaces with a 0.3 M NaOH solution in nuclease free water. Immediately rinse surfaces with nuclease free water and wipe dry with RNase free wipes.
Place a Whatman Benchkote Surface Protector on the primary work surface followed by an RNase free 16.4"x14.43" Kimwipe sheet.
Reserve a centrifuge. Clean surfaces with RNase-zap and rinse with nuclease free water.
Keep a sealed Uline box of kits, reagents, pipettes, and consumables used for RNA work.
Keep a sealed box of RNA work reagents in the −80°C, −20°C, and 4°C freezers and fridge.
Prepare 1.5 mL tubes
Prepare and label six sets of 1.5 mL nuclease-free Eppendorf tubes per sample.
You will need a new set of tubes for steps:
1 - Counting
5 - Lysate
10 - 1st Elution
18 - 2nd Elution
20 - Purified RNA
22 - QC aliquot
Label ZymoBIOMICS columns
Label one set of ZR BashingBead Lysis Tubes.
Label two sets of green Zymo-Spin IICG columns per sample and place the columns in a Collection Tube.
Label one set of Zymo-Spin III-HRC Filter columns per sample and place the columns in a Collection Tube.
Prepare DNA digestion solutions
DNase is supplied by Zymo as a lyophilized pellet.
Reconstitute DNase I (#E1009-A) in 275 µL of RNase- free H2O. Mix by gentle inversion. DNase is denatured by vortexing.
Store aliquots of DNAse at -20 °C
Determine the volume of DNase required for DNA digestion.
For each sample, 5 µL of DNase and 10 µL of DNA Digestion Buffer is needed.
Thaw DNase on ice. Add DNase to the DNA Digestion Buffer right before DNA digestion in step 12.
Add 96 mL 100% EtOH to the RNA Wash Buffer concentrate.
Check the box to confirm you have added EtOH. Write your initials and the date. Cover your markings with clean and clear scotch tape to avoid EtOH from dissolving the marks.
Prepare the Qubit reagents
Store Qubit RNA BR Reagent (component A) at room temperature in the dark.
Store Qubit RNA BR Buffer (component B) below 30 °C
Store Qubit BR Standard #1 (component C) and Qubit BR Standard 2 (component D) at 2 °C - 8 °C
Prepare the Bioanalyzer reagents
Store the Agilent RNA 6000 Pico Chips at room temperature.
Store the RNA 6000 Pico Marker at 4 °C
Store the RNA 6000 Pico Conditioning Solution at 4 °C
Store the RNA6000 Pico Gel Matrix at 4 °C
Protect all dyes and dye mixtures from light.
Prepare the Bioanalyzer gel
Place a clean spin filter into a 1.5 mL Eppendorf tube.
Add 550 µL of RNA gel matrix into a spin filter.
Centrifuge at 1,500 ×g for 10 min at room temperature.
Aliquot 65 µL of filtered gel into nuclease free 1.5 mL tubes and store aliquots at 4 °C
Use filtered gels within 4 weeks.
Set up heating program on the BioRad T100 Thermal Cycler
Set up a program that heats a sample to 70 °C for 2 min.
Prepare the Bioanalyzer RNA ladder
Thaw the ladder if previously frozen.
Centrifuge the ladder for 30 s at 10,000 ×g, room temperature.
Incubate the ladder at 70 °C for 2 min on a clean heating block.
Immediately place the ladder on wet ice for 1 min.
Add 90 µL of nuclease free water and invert to mix.
Make 3 µL aliquots and store at -80 °C
Replacing and Cleaning the Bioanalyzer RNA Electrode Cartridge
To remove the electrode cartridge, pull on the cartridge release latch (see image below) on the bottom of the lid. The cartridge should slide outwards.
Replace the electrode cartridge on the Bioanalyzer with a 16 pin bayonet RNA electrode cartridge dedicated for RNA use only.
When not in use, store the dedicated RNA electrode cartridge safely in a labeled box and replace it with a DNA/protein cartridge.
Agilent 2100 Bioanalyzer
Fill an electrode cleaning chip with 350 µL of nuclease free H2O.
Place the electrode cleaning chip into the Bioanalyzer and close the lid gently. Wait 5 min.
Open the lid and remove the electrode cleaning chip. Leave the lid open for 30s to allow the water to evaporate off the electrodes.
Inspect the electrodes for bubbles or drops of water. Residual water left on the electrodes will cause the run to fail and may introduce artifacts. In many cases, the chip will not pass the initial preparation step of data acquisition.
Power on the Bioanalyzer Instrument
The Agilent 2100 Bioanalyzer is an old instrument and will often have trouble at start-up.
Primed chips are good for 5 min, so it is imperative that both the instrument and software are ready.
Make sure that the computer is powered on and the software is opened before powering on the instrument.
If the instrument is successfully connected and recognized by the software, the Home Screen should appear like the image below. Everything in the start run checklist should have a green check mark except for the detection of the chip.
Software Home Screen upon launch. System status is displayed to the right.
Set up the Chip Priming Station
Ensure that the base plate is set to position C. If not, use a PH0 Phillips head screw driver to loosen the screw on the underside of the base plate. Adjust the plate to position C and then tighten the screw to secure the plate to the priming station.
Please read step 42 to see a detailed diagram of the chip priming station.
A syringe is supplied with every new RNA 6000 Pico kit and must be replaced with each kit.
Ensure that the syringe is tightly secured to the Luer lock adapter on the chip priming station. Twist the syringe barrel clockwise to lock. Ensure that the white rubber gasket that meets the chip is in good condition.
Test the syringe and gaskets by depressing the plunger on a used chip and observe the resistance. If the plunger seal fails to retract past the 0.3 mL mark, the seals may be compromised.
RNA extraction and purification
RNA extraction and purification
Determine the cell density of your sample. See (Camacho & Merchant, 2024) protocol for counting cells with a hemocytometer.
Calculate the volume of culture required to collect 1–3x108 cells.
Transfer the required volume of culture to a 15 mL high speed centrifuge tube.
Centrifuge for 2 min at 10,000 ×g, room temperature, and pour off supernatant.
Note
Use the tubes specified in the materials section, ensuring that they are rated to withstand a centrifugal force of 10,000 ×g.
Resuspend pellet in 750 µL DNA/RNA Shield solution by pipetting up and down, then transfer the solution to a ZR BashingBead Lysis Tube (0.1 & 0.5 mm) and cap tightly.
Use the Biospec Mini Bead Beater 16 to lyse the cells for 3 min and 30 s in the 4 °C cold room. Repeat for a total of 7 min.
Centrifuge the lysate for 30 s, 16,000 ×g, at room temperature, and transfer 400 µL of the supernatant into a new nuclease-free 1.5 mL tube.
Add 400 µL of RNA Lysis Buffer to the supernatant and mix.
Add 800 µL of 100% ethanol to the sample and mix.
Place a green Zmyo-Spin IICG Column into a Collection Tube and transfer 750 µL of the mixture into the column. Centrifuge for 30 s, 16,000 ×g, at room temperature. Discard the flow-through and repeat until all of the mixture from step 7 has been run through the column.
Add 400 µL RNA Prep Buffer to the column and centrifuge for 30 s, 16,000 ×g, at room temperature. Discard the flow-through.
Verify that ethanol has been added to the RNA Wash Buffer.
Add 400 µL RNA Wash Buffer to the column.
Centrifuge for 30 s, 16,000 ×g, at room temperature.
Discard flow-through.
Centrifuge again for 30 s, 16,000 ×g, at room temperature to remove residual liquid.
Transfer the column to a new nuclease-free 1.5 mL tube.
Add 85 µL of RNase free water directly to the center of the column matrix.
Do not add water to the walls of the tube.
Incubate the column for 1 min at room temperature.
Centrifuge for 30 s, 16,000 ×g, at room temperature.
Optional:
Perform a second elution into a separate 1.5 mL tube by adding an additional 85 µL of RNase free water directly to the center of the column matrix.
Incubate the column for 1 min at room temperature.
Centrifuge for 30 s, 16,000 ×g, at room temperature.
Determine the concentration of the second eluted sample using a NanoDrop.
Discard the column.
See the "Before Start" section for notes on DNase preparation.
In a new 1.5 mL tube, add the required volumes of DNA Digestion Buffer and DNase I.
For each sample, add 5 µL of DNase I to 10 µL of DNA Digestion Buffer.
Add 15 µL of the mixture to each sample.
Incubate the samples at room temperature for 15 min.
Optional: While you wait, prepare the Zymo-Spin III-HRC Filter in step 20. Set the filter aside until you reach step 20, or store frozen.
Add 200 µL of RNA Lysis Buffer to the sample and mix.
Add 300 µL of 100% ethanol and mix.
Place a green Zymo-Spin IICG Column into a Collection Tube and transfer the mixture into the column. Centrifuge for 30 s, 16,000 ×g at room temperature. Discard the flow-through and repeat until all of the mixture from step 14 has been run through the column.
Add 400 µL RNA Prep Buffer to the column and centrifuge for 30 s, 16,000 ×g at room temperature. Discard the flow-through.
Add 700 µL RNA Wash Buffer to the column.
Centrifuge for 30 s, 16,000 ×g, at room temperature.
Discard flow-through.
Add 400 µL RNA Wash Buffer to the column.
Centrifuge for 30 s, 16,000 ×g, at room temperature.
Discard flow-through.
Centrifuge again for 30 s, 16,000 ×g, at room temperature to remove residual liquid.
Transfer the column to a new nuclease-free 1.5 mL tube.
Add 50 µL – 100 µL of RNase free water directly to the center of the column matrix.
Do not add water to the walls of the tube.
Incubate the column for 1 min at room temperature.
Centrifuge for 30 s, 16,000 ×g, at room temperature.
Optional:
Perform a second elution into a separate 1.5 mL tube by adding an additional 50 µL to 85 µL of RNase free water directly to the center of the column matrix.
Incubate the column for 1 min at room temperature.
Centrifuge for 30 s, 16,000 ×g, at room temperature.
Determine the concentration of the second eluted sample using a NanoDrop.
Discard the column.
Place a Zymo-Spin III-HRC Filter into a new Collection Tube.
Add 600 µL of ZymoBIOMICS Prep Solution. Please note this is different from the RNA Prep Buffer. Centrifuge for 3 min, at 8,000 ×g, at room temperature.
Discard the flow-through and collection tube and transfer the Zymo-Spin III-HRC Filter into a new 1.5 mL tube.
Transfer the eluted RNA from step 19 into the prepared Zymo-Spin III-HRC Filter and 1.5 mL tube from step 20.
Centrifuge for 3 min at 16,000 ×g, room temperature.
Aliquot 5 µL of purified RNA into a new 1.5 mL tube.
This aliquot will be used for QC analysis and avoids freeze thaw cycles of the rest of the purified sample.
1.5 µL will be used for NanoDrop quantification, A230/A260, and A280/A260 ratio values.
1 µL will be used for Qubit fluorescence quantification.
1 µL will be used for quality assessment using a Bioanalyzer.
1.5 µL is reserved for pipette error or re-measurement/ re-assessment.
Place the QC RNA sample on wet ice.
Flash freeze the RNA samples (not QC samples) in liquid nitrogen and store at -80 °C .
Measurement of RNA concentration
Measurement of RNA concentration
Check the RNA concentration, A260/A230, and A260/A280 ratio values using the NanoDrop.
We recommend using 1.5 µL of blank and sample solutions.
Note
The typical RNA concentration extracted from 108 cells and measured with a NanoDrop ranges between 150–400 ng/µL. Very pure RNA will have an A260/A280 ratio of ~2.1 and an A260/A230 ratio above 2.0. A260/A280 values below 1.8 may indicate potential DNA or protein contamination. Low A260/A230 values typically indicate contamination by wash buffers, organic compounds, guanidine salts, or phenol.
Bring the Qubit BR standards #1 (component c) and #2 (component d) to room temperature.
It is very important that all solutions are at room temperature.
Protect the solutions from light.
If you plan to run the Bioanalyzer QC immediately after Qubit RNA quantification, bring the Bioanalyzer reagents to room temperature as well.
Pour out enough thin-wall, clear 0.5 mL Qubit Assay Tubes for all your samples (with replicate measurements), plus two additional tubes for standards.
Label the top of the lids. Do not label the side of the tubes. Marking the side of the tube may interfere with the light path of the fluorometer.
Calculate the volumes required to make a working solution containing RNA BR Reagent 1:200 in RNA BR Buffer.
For every sample you will add 199 µL of the working solution to a tube and 1 µL of RNA sample.
You will measure two standards that will each require 190 µL of working solution and 10 µL of the corresponding standard. We recommend measuring each RNA sample with two technical replicates.
For example, if you are measuring 4 samples, make a working solution for 10 reactions (4 samples x 2 replicates each sample, plus two standards).
200 µL x 10 reactions = 2 mL working solution required (see note).
1:200 RNA BR Reagent:RNA BR Buffer = 10 µL RNA BR Reagent + 1990 µL RNA Buffer
Note
There will be 22 µL of surplus working solution so it is not necessary to make an extra reaction to compensate for spillage and pipetting error.
Mix the working solution in a nuclease free plastic container.
Do not use a glass container.
Protect the working solution from light.
For each sample, add 199 µL of working solution and 1 µL of RNA sample to a thin-wall, clear 0.5 mL Qubit Assay Tube.
For each standard, add 190 µL of working solution and 10 µL of the corresponding standard.
Invert and flick all tubes to mix and centrifuge lightly to pull all liquid to the bottom of the tube.
Incubate the tubes upright, in the dark, and at room temperature for 2 min. The reactions are stable for up to 3 h, but we recommend reading them immediately after the 2 min incubation.
Plug the Qubit instrument into an outlet to power on the instrument. A power button does not exist on the Qubit 4 instrument.
On the Home screen, select "RNA". See panel a in the image below.
Select "RNA Broad Range". See panel b in the image below.
Select "Read Standards". See panel c in the image below.
Make sure to wipe the sides of all tubes with a clean Kimwipe.
Insert the tube containing Standard #1.
Close the lid and select "Read standard". See panel d in the image above.
Record the fluorescence value (RFU) associated with the standard's concentration. See panel e in the image above.
Remove the tube.
Insert the tube containing Standard #2.
Close the lid and select "Read standard".
Record the fluorescence value associated with the standard's concentration. See panel f in the image above.
Remove the tube.
Note
The raw fluorescence values (RFU) are important for extrapolating concentrations of samples that are out of range. This extrapolation should only serve to estimate a dilution factor for the sample. A second reading of the diluted sample is necessary to obtain an accurate RNA concentration. See the note in step 34. Depending on the purity of the extracted RNA, Qubit concentrations are usually about 60%–95% of the RNA concentrations determined by a NanoDrop. The RNA broad range assay kit has a linear range of quantitation between 20–1000 ng/µL. Dilute your samples accordingly.
Select "Run samples" and input 1 µL for the "Sample Volume" option and ng/µL for the "Unit" option. See panel g in the image on step 31.
Insert a sample, close the lid, and select "Read tube". Record the concentration.
Remove the tube and repeat until all tubes have been read.
Note
If a sample reads "Out of Range", select data, select the sample, and record the raw fluorescence value (RFU).
Plot the raw fluorescence values of the standards on the x-axis and the concentration of the standards on the y-axis. Draw a line of best fit between the standards and determine the slope and y-intercept. To estimate the concentration of a sample that is out of range, plot the raw fluorescence value on the line or use the equation y=mx+b.
Determine the dilution factor needed to dilute the RNA concentration so that it is within range.
Dilute the RNA sample in water, do not dilute the reaction. Remake the reactions with new standards and measure the standards with the diluted samples.
Return the standards to the 4 °C referigerator.
Assessment of RNA quality
Assessment of RNA quality
Allow all reagents to equilibrate at room temperature for at least 30 min.
Protect all solutions from light.
Keep samples on wet ice.
While reagents equilibrate to room temperature, follow steps 37 and 38 to dilute and denature RNA samples.
The required RNA input for the RNA Pico Kit on the Agilent 2100 Bioanalyzer is 200–5000 pg in one µL. We recommend using 5000 pg of RNA.
It is difficult to obtain only 5000 pg of RNA in one µL so you will need to dilute into a larger volume.
For example, if you have an RNA concentration of 1000 ng/ µL then you should add 0.5 µL of RNA sample to 99.5 µL of nuclease free water.
Add 5 µL of the diluted sample to a nuclease free 0.2 mL PCR tube.
Denature the diluted RNA samples at 70 °C for 2 min (see the "Before Start" section for set up).
You do not need to denature the RNA ladder, if it has been denatured before.
Once all reagents are equilibrated to room temperature, vortex the RNA dye concentrate for 10 s and centrifuge for 30 s, 16,000 ×g at room temperature.
Add 1 µL of RNA dye concentrate to a 65 µL aliquot of filtered gel from the "Before Start" section.
Vortex the solution for 5 s and centrifuge for 10 min at 13,000 ×g, room temperature.
The prepared gel must be used within one day. One aliquot is enough for 2 chips.
Put a new chip into the chip priming station.
Add 9 µL of gel-dye mix into the well marked G (in a black circle).
Read this entire step before attempting. See the "Before Start" section for setting up the chip priming station.
Keep a timer in sight with seconds displayed. You will need to depress the plunger for exactly 30 s in this step.
Pull the plunger back so that the plunger seal is at the 1 mL position.
Close the chip priming station and listen for the latch to click.
Press the plunger down until the plunger flange is locked by the syringe clip.
Wait exactly 30 s then release the plunger by squeezing the syringe clip lever.
Wait 5 s and watch the plunger slowly rise until the plunger seal is around the 0.7 mL mark.
Slowly pull the plunger flange up until the plunger seal is back to the 1 mL position, moving at about 0.05 mL / s.
Bioanalyzer chip priming station.
Note
If the plunger fails to rise above the 0.3 mL mark after releasing the syringe clip lever, then it is possible that the gasket has a leak or the syringe was not securely tightened to the Luer lock adapter of the chip priming station.
You will have to prime another chip.
Open the chip priming station and pipette 9 µL of gel-dye mix into the other two wells marked G.
Add 9 µL of the RNA conditioning solution into the well marked CS.
Add 5 µL of RNA marker into all 11 sample wells and the well for the ladder.
Add 1 µL of the RNA ladder to the well marked for the ladder.
Add 1 µL of diluted RNA sample to each sample well.
Add1 µL of RNA marker to each unused sample well.
Insert the front of the chip at an angle and press it against the spring-loaded latch.
Slowly lower the back end of the chip so that it is horizontally level and held in place by a spring-loaded latch.
Press the start button to vortex for 1 min at 2400 rpm (max speed). The vortex will automatically stop.
IKA Vortexer: The blue arrow shows the chip insertion direction. Insert the front of the chip at an angle, press it against the spring-loaded latch, lower the back slowly, and ensure it sits level once fully secured. The chip will fly off the vortexer if it is not secured.
See the "Before Start" section for instrument start up.
Place the chip into the instrument.
The status on the computer screen should change to signify that the chip has been detected.
Now, you may change the names of the samples to your desired sample names.
Press start.
The instrument will take a few minutes in a preparation phase.
This is normal. An electropherogram with no data will be displayed.
A progress bar indicating the progress of the preparation step will be displayed at the bottom right of the screen. This progress bar will reset for each well during the analysis of each sample.
While the instrument prepares for the analysis, store the reagents in their designated locations at the specified temperatures.
When the run is finished, export the data as .bmp images of individual electropherogram, .csv files of the fluorescence values over time, .cdf, .pdf, and .xad files.
Electropherogram of high quality RNA extracted using this protocol are shown below for photoautotrophic and mixotrophic cells. An extra peak representing chloroplastic rRNA is present in the electropherograms of RNA extracted from photoautotrophic cells.
Electropherograms of high quality RNA extracted from UTEX 250 grown in photoautotrophic (top) and mixotrophic (bottom) conditions.
An example of moderately degraded RNA extracted using another protocol is shown below for reference.
Remove the chip from the instrument.
Clean the electrodes by filling the electrode cleaning chip with 350 µL of nuclease free water.
Close the lid to soak the electrodes for 5 min.
Remove the electrode cleaning chip.
Replace the dedicated RNA cartridge with a DNA/ protein cartridge.
Store the RNA cartridge safely in a sealed box. Make sure that the electrodes are facing upwards.
Shut the instrument down.
Protocol references
Dimitrios Camacho, Sabeeha Merchant 2024. Determination of Auxenochlorella protothecoides Cell Density With a Hemocytometer. protocols.iohttps://dx.doi.org/10.17504/protocols.io.rm7vzjk12lx1/v1
Acknowledgements
We extend our sincere thanks to Sunnyjoy Dupuis, Dr. Anne Glaesener, and Dr. Sean Gallaher for their suggestions, insights, guidance, and feedback on the RNA extraction protocols. We are also deeply grateful to Dr. Daniela Strenkert for her expert guidance and instruction on operating the Qubit fluorometer and Bioanalyzer.