RT-qPCR

Isolation of RNA from cultured cells

Goal: Isolate RNA from cultured primary cells for subsequent reverse transcription and quantification. RNA should be of high quality and also of sufficient quantity and concentration that 1 mg of RNA can be used as a template for the subsequent reverse transcription step. In theory, variability in the amount of template RNA should be accounted for during the qPCR step, but we find it preferable to standardize the input in order to remove as many variables between conditions as possible. We chose 1 mg as an arbitrary standard, but one could choose their own standard within the input range recommended for SuperScript IV reverse transcription (10 pg - 5 mg). We generally find that 12-well culture plates containing cells approaching confluency provides sufficient RNA, but you may need to scale up (or be able to scale down) depending on your cell type.

We use the Qiagen RNEasy Mini kit (74106) and follow their protocol (https://www.qiagen.com/us/resources/download.aspx?id=0e32fbb1-c307-4603-ac81-a5e98490ed23&lang=en), with additional details provided below.

1. Rinse cells 1x with 1 ml/well room temperature DPBS and aspirate with a vacuum trap. Scale volume up or down depending on your culture vessel.

2. Lyse cells directly in the plate with 350 ul RLT buffer containing 10 ul/ml 2-mercaptoethanol.

3. Gently pipet the volume in and out several times in each well to ensure lysis of cells. Try not to introduce bubbles. Continue until there are no visible clumps or sheets (this is generally only seen if cells are confluent or overgrown).

4. Add 350 ul 70% ethanol and mix by pipetting.

5. Transfer to RNEasy Mini spin column and process according to Qiagen RNEasy mini kit quick-protocol steps 3-7. Briefly, this involves spinning the lysis solution through the column, followed by a 700 ul wash with RW1, two 500 ul washes with RPE, a drying step, and elution with RNase-free water. Lysis, RW1, and the first RPE spins are 15s at >8000g in a tabletop centrifuge, the second RPE spin and drying spin are two minutes, and the elution spin is one minute.

Note: We omit the optional DNase digestion. A 260/280 ratio close to 1.8 (see below) may indicate DNA contamination and suggest inclusion of this step. We recommend designing primers for the qPCR step that span introns, so that any residual DNA will not contribute significantly to the qPCR signal. If this is not possible (e.g. interest in a single-exon gene), it may be worthwhile to include this step. 

Note: We include the optional membrane-drying spin.

Note: We elute in a single elution of 30 ul RNase-free water, to maximize RNA concentration.

6. Place tubes on ice and quantify the RNA concentration using 1 ul on a Nanodrop. Ensure the concentration of all samples is high enough for the subsequent reverse transcription step (e.g. it must be > 90.9 ng/ml to fit 1 mg in 11 ul). Check the 260/280 ratio, which should be close to 2.0 for RNA. More information can be found here: https://tools.thermofisher.com/content/sfs/brochures/T123-NanoDrop-Lite-Interpretation-of-Nucleic-Acid-260-280-Ratios.pdf

Note: If the RNA will be used for an especially sensitive application, such as library preparation for RNA sequencing, quantification and quality analysis using a TapeStation is preferred to a Nanodrop.

7. RNA can be used immediately for reverse transcription or stored at -70 °C or -80 °C.

Reverse Transcription

Goal: Reverse transcribe RNA into cDNA for quantification using qPCR. We utilize a mix of random hexamer primers (which bind complementary sequences across all RNA, including the coding portions of mRNA) and oligo(dT) primers (which bind the poly(A) tail of mRNA) in a molar ratio of 2:1 random hexamer:oligo(dT). This can be switched to all random hexamer, all oligo(dT), or sequence specific primers depending on the application. We have also had success using the SuperScript IV VILO reagent which includes primers designed for qPCR already in the master mix, but did not use that particular reagent in Dinsmore and Soriano, eLife, 2022.

We follow the protocol for SuperScript IV from ThermoFisher (https://tools.thermofisher.com/content/sfs/manuals/SSIV_Reverse_Transcriptase_UG.pdf), with additional details provided below.

1. Thaw all reagents on ice.

2. Mix 1 mg RNA, 1 ul 10 mM dNTP mix, 1 ul primer mix, and RNase-free water to bring the volume to 11 ul.

Note: The protocol recommends 1 ul of 50 mM primer, so we make a 2:1 mix of 50 mM randomer hexamer (ThermoFisher, 48190011, stock concentration 1.515 M diluted to 50 mM):50 mM oligo(dT) (ThermoFisher, 18418012, stock concentration estimated at 101 mM, diluted to 50 mM) and use 1 ul of this 2:1 mix at this step.

 Note: For multiple samples, we assemble the reactions in 8-well PCR strip tubes and perform all the incubation steps using a thermocycler.

 Note: It is recommended to include a no-template control.

 Note: The amount of input RNA chosen can be anywhere from 10 pg-5 mg.

3. Mix, spin down using a microfuge with strip tube attachment.

4. Incubate 5 min at 65 °C.

5. Place on ice 1 min.

6. Add 4 ul 5x SSIV buffer, 1 ul 100 mM DTT, 1 ul RNaseOUT, 1 ul SSIV.

Note: We make a single mix of the above reagents with volumes multiplied based on the number of samples plus 15% to account for loss during pipetting, then add 7 ul to each sample.

7. Briefly vortex and spin down as in step 3.

8. Incubate 10 min at 23 °C, 10 min at 55 °C, 10 min at 80 °C.

9. Place on ice.

10. Add 80 ul water to dilute cDNA volume to 100 ul.

Note: This is optional but based on the amount of input RNA, the cDNA remains at a sufficiently high concentration for qPCR. This step can be omitted or adjusted based on your own results, input RNA, etc.

Note: Any nucleic acid and nuclease-free water source, such as MilliQ water, is acceptable. We use the RNase-free water included with the SuperScript kit for convenience. 

11. cDNA can be used immediately for qPCR or stored at -20 °C.

qPCR

Goal: Accurate quantification of gene expression levels across different samples. We use NEB Luna 2x qPCR Master Mix in 96-well plates on a BioRad iQ5 qPCR Thermocycler. We design our primers, whenever possible, to span introns, thereby increasing specificity for cDNA originating from mRNA and reducing signal from any contaminating genomic DNA. We use a single gene for normalization of samples, but if there is space to include multiple genes for normalization (such as on a 384-well plate) this will provide more accurate results. An appropriate normalization gene (or genes) can be chosen by testing multiple candidate genes across several samples (making sure to include samples from the different conditions relevant to the experiment such as genotype, growth factor stimulation, etc.) and normalizing every candidate gene to every other candidate gene. The pairs with the lowest variance across samples are most appropriate for use in normalization. If multiple control genes are included in the final experiment, the geometric mean of the multiple control genes is used for normalization. In Dinsmore and Soriano, eLife, 2022, Hprt was used as the single gene for normalization.

We follow the NEB Luna Master Mix protocol (https://www.neb.com/protocols/2016/11/08/luna-universal-qpcr-master-mix-protocol-m30030), with additional details provided below.

1. Thaw all reagents on ice.

Note: Primers are ordered from IDT, resuspended at 100 mM in 0.1x TE buffer (1 mM Tris-HCl pH 8.0, 0.1 mM EDTA), and then used to make a 10 mM working dilution in water. Both are stored at -20 °C and the working dilution is used for setting up qPCR reactions. The stock is only thawed to make more working dilution.

2. Calculate total number of reactions for each cDNA and primer set. We run three technical repeats for each cDNA-primer set pair, arranged side-by-side on 96-well plates. More technical replicates can be run if there is room. For instance, 2x2 squares of four repeats fit well on 384-well plates. When calculating volumes, include an extra 15% to account for loss during pipetting. 

3. Assemble two sets of master mixes: one for each cDNA sample containing sufficient volume (e.g. # genes * 3 * 1.15) and one for each gene containing the forward and reverse primers.

4. Add 1 ul of primer mix to the bottom of each well.

5 Add 19 ul of cDNA mix along the side of each well.

Note: for steps 2 and 3, we find it easiest to arrange the plate so that each column contains a single primer pair and each row contains a single cDNA. (e.g. columns 2-4 contain gene 1, columns 5-7 contain gene 2; row B contains sample 1, row C contains sample 2, etc). Strip tubes containing the appropriate primers or cDNA mix can be arranged along each side and the volumes added to the wells rapidly using multichannel pipets.

Note: Make sure the drop of primer is deposited in the bottom of each well and drag the pipet tips out along one side of the well. Then, deposit the cDNA along the opposite side of the well and drag out. If this rule is followed, the same pipet tips can be used for depositing cDNA mix in every well as there will be no cross-contamination of primer. If the tip is accidentally inserted too far into the well and touches the primer, it should be discarded for subsequent pipetting.

Note: Wells at the edges of the plate are subject to evaporation due to warping of the plate and seal and should therefore be avoided if possible. If these wells are used, plates should be visually inspected after the qPCR-step for volume loss and data manually inspected for inconsistent results. These data should not be used if either are discovered. Your results may vary by machine, plate, and seal.

6. Seal the top of the plate with transparent film. Carefully align the film and use a roller or straight edge to slowly and evenly apply the seal, avoiding wrinkles and bubbles. Once applied, use a straight edge to apply additional pressure at the corners and along the edges to prevent evaporation.

7. Spin the plates 1 minute at 1000 rpm in a swinging bucket centrifuge at room temperature to ensure material is mixed and collected at the bottom of each well.

8. Run the plate according to the following cycling conditions.

1 min 95 °C

15 sec 95 °C

30 sec 60 °C

Repeat steps 2-3 for 39 (40 total) cycles

Melt curve (can be added in qPCR machine software).

9. Save the data. The plate can be stored at 4 °C (a few days) or -20 °C (longer), run out on an agarose gel for band analysis, or discarded.

10. Inspect the melt curves for each primer-pair to see a single curve across the sample. This can be compared to simulated curves (https://www.dna-utah.org/umelt/quartz/um.php). Samples can be checked on an agarose gel that a single band of the expected size was amplified. If there are multiple bands or inconsistent melt curves, new primers should be designed. More information is available here: https://www.idtdna.com/pages/education/decoded/article/interpreting-melt-curves-an-indicator-not-a-diagnosis

11. Average the Ct values across the three (or more) technical repeats for each cDNA-gene pair.

12. Calculate the relative expression level using the DDCt method (Livak and Schmittgen, 2001).

13. Perform statistical analysis, such as t-tests, to compare differences between control and treatment samples for significance. This is greatly facilitated using software such as Graphpad Prism, which can perform correction for multiple testing (e.g. testing 20 genes at once, you would expect one to be significantly different purely by chance using a 0.05 significance threshold).