Single Cell Quantitative Polymer Chain Reaction (sc-qpcr)

Single Cell Quantitative Polymer Chain Reaction (sc-qpcr) Analyzing gene expression profiles from a bulk population of cells provides an average profile which may obscure important biological differences in individual cells. Using an optimized workflow for qrt-pcr, gene expression profiles of single cells, embryos and blastomeres can reveal distinct gene expression profiles for individual cells, and a large expression level range of almost every gene. Importantly, this technique allows for the identification and characterization of small subpopulations. Figure1: Schematic Workflow of Cell-to-CT Experiment. The single Cell-to-CT kit workflow is comprised of 4 functional steps. (1) Cell Lysis. (2) Reverse Transcription. (3) cdna Pre-Amplification. (4) Real Time PCR. (From: Cell-to-CT Kit Manual, Ambion LifeTec 2010) REFERENCES: Abruzzese et al (2013) Single Cell Gene Expression Analysis of Pluripotent Stem Cells. Pluripotent Stem Cells. Vol 997 pg 217-224 1

REAGENTS: Required Material Source Storage PBS (Ca 2+ and Mg 2+ Free) Gibco: Cat#14190-144 RT Tris EDTA Buffer ph8.0 (Nuclease Free) Ambion: Cat#AM9858 RT Nuclease Free Water Ambion: Cat#AM9937 RT SSO Advanced RT-PCR Master Mix Biorad: Cat# 1725275-20 o C Cell-to-CT Kit Contents: Ambion PN4458236, 400 rxt Reagent Volume (µl) Storage Single Cell Lysis Solution 4.0mL 4 o C Single Cell DNASE 200µL -20 o C Single Cell STOP 400µL -20 o C Single Cell VILO RT Mix 1.2mL -20 o C Single Cell Superscript RT 600µL -20 o C Single Cell PreAmp Mix 2.1mL -20 o C Taqman Gene Expression MM 5.0mL 4 o C Prepare Reagents: 1. Single Cell Lysis Solution: Arrives frozen along with all other reagents. Simply move this reagent to an RNA protected 4 o C fridge. 2. Single Cell DNASE: This reagent has been confirmed as identical to Ambion AM2222. 3. Single Cell VILO RT Mix: For the sake of convenience, once opened, we split this reagent into 25uL aliquots. A single reaction needs 3µL, and we typically prepare samples in an 8-strip tube. This is done to avoid unnecessary freeze-thaw. Simply remove aliquots in increments of 8x samples. 4. Single Cell PreAmp Mix: This item takes an unusually long time to thaw. Do thaw on ice but begin thawing this item at the beginning of the protocol to allow sufficient time to reach a liquid phase. Do not attempt to hasten the thawing process. 5. Taqman Gene Expression MM: Although this item is included in the kit, this protocol does not mention it because our lab uses SYBR based qpcr technology rather than probe based to be cost effective as well as being able to test multiple primers at a time. (Some of our targets are intentionally within highly repetitive regions). 2

Single Cell Embryo-to-CT Protocol SAMPLE COLLECTION: Embryo Collection: Embryos: Please refer to EMBRYO COLLECTION PROTOCOL. Docx. In addition to normal washes, add a final wash into a PBS (ph 8.0) droplet. Cull Culture: Please refer to commercial protocol for collection method. In Brief, this will include trypsinization followed by either mouth pipetting or Flow Sorting. SINGLE CELL LYSIS/DNASE I TREATMENT: NOTE: This is double the volume from the original AMBION protocol. The reason for this is that these embryos are much larger than the 1-10 cultured cells the protocol author had in mind. In fact, a mouse oocyte volume is approximately 30x the volume of a MEF cell. Therefore, we may already be reaching saturation with just a single embryo! 1. Create Lysis/DNASE Solution and keep Master Mix on ICE until needed: Component Volume per Reaction (1X) Volume Per Reaction (8x) Single Cell/Lysis Solution 18 ul 144 ul Single Cell DNASE I 2 ul 16 ul Total 20 ul 160 ul 1. To an 8-well strip of PCR tubes, directly add a single embryo to each well from PBS Droplet. 2. To do this, set pipette to 1uL or less (remember to keep this volume standard for all collections!) 3. Be sure to use fine tip pipettes, the kind used to load PAGE Gels (Fisher: 02-707-81). Normal pipettes may work, but they are difficult to use under the microscope. Carefully view pipette tip under Stereo-microscope to ensure only a single good looking embryo is selected. 4. To make sure the embryo is transferred, push the pipette plunger until a bubble is released. IMPORTANT: Do not add more than 1uL of PBS from droplet along with Embryo. RNASE inhibitors in Lysis Mix may not work! Place strip on ICE until ready for next step! 5. Add 20 ul of Lysis/DNASE Solution to each tube of 8 well strip VERY quickly. 6. Remove tubes from ice and flick tubes very gently and spin down VERY briefly. 7. Incubate at Room Temperature (RT. Approximately 20-23.5 o C) for 10 minutes. This step is incredibly important, as we need to make sure the DNASE degrades as much DNA as possible, since some of the Retrotransposons we are assaying may exist in thousands of genomic copies! 8. Do NOT exceed 30 minutes at RT, as DNASE will display RNASE activity. If you must exceed 30 minutes, place samples on ICE. 9. Add 2uL of Single Cell Stop Solution directly to each sample. Do NOT add to the side of the tube. Instead, touch the pipette tip to the surface of the 21uL reaction. 10. To ensure proper Stop Solution concentration, pipette up and down 5 times. 11. Total Volume at this point: 23uL. OPTIONAL: Stop point. Samples can be frozen at -20 o C or -80 o C. The AMBION protocol states the samples can be freeze/thawed up to five times at this point with no problems. However, we try to do the experiment without freezing even once. 3

OPTIONAL RT+ and RT- CONTROL NOTE: This is a MAJOR deviation from the Ambion Protocol. We find that since Embryos have much more RNA than the samples typically associated with this protocol, there is much more material than needed per reaction. We highly recommend doing this at least for the initial assays to ensure the experiment is working properly and the DNASE I treatment is effective. If you find there is extensive amplification in the -RT samples, then add an additional 5 minutes to the initial Lysis/DNASE I incubation. If you chose to NOT to do the RT- control, skip to REVERSE TRANSCRIPTION 1. Move 11.5 ul (half of the volume) to a properly labeled 8-well strip (New RT- Controls). 2. Use strip 1 as an RT+ sample and strip 2 as RT- sample. 3. Prepare RT Reaction Mix: Component Volume per Reaction (1X) Volume Per Reaction (8x) Single Cell VILO RT Mix 3 ul 24 ul Single Cell SuperScript RT 1.5 ul 12 ul Total 4.5 ul 36 ul 4. Add 4.5 ul of RT Reaction Mix directly to each RT+ sample. Mixing is not necessary. 5. Add 4.5 ul of Tris-EDTA ph 8.0 (TE Buffer) to each RT- Sample. Mixing is not required. 6. Place samples into PCR Thermocycler and run the following Protocol: 7. At this point, the sample volume is 16 ul. 8. Skip to PREAMPLIFICATION! Temp Time 25 o C 10 min 42 o C 60 min 85 o C 5 min Optional: Stop point. Samples can be stored at -20 o C. Ambion states these samples can now be freeze/thawed 5 times without any problems. 4

REVERSE TRANSCRIPTION: NOTE: This part of the protocol only works if you did NOT do the RT- Control section above. 1. Prepare RT Reaction Mix: Component Volume per Reaction (1X) Volume Per Reaction (8x) Single Cell VILO RT Mix 6 ul 48 ul Single Cell SuperScript RT 3 ul 24 ul Total 9 ul 72 ul 2. Add 9 ul of RT Reaction Mix to each sample. Mixing is not necessary. NOTE: This is STILL twice the normal Ambion Protocol volume. 3. Place samples into PCR Thermocycler and run the following Protocol: 4. At this point, the sample volume is 32 ul. Temp Time 25 o C 10 min 42 o C 60 min 85 o C 5 min Optional: Stop point. Samples can be stored at -20 o C. Ambion states these samples can now be freeze/thawed 5 times without any problems. SPLITTING SAMPLE VOLUME: NOTE: This is a MAJOR deviation from the Ambion Protocol. However, since there is so much material per embryo, we find that we can extend the usefulness of each embryo by splitting the reaction at this point. The next step forces you to choose your genes of interest, which can NOT be later modified, we feel this addition to the protocol can provide valuable technical replicates to each sample and well as assaying additional gene from the same embryo after the initial experiment. 1. Move 16 ul (half of the volume) to a properly labeled 8-well strip. NOTE: Even if you chose to do the RT- control, all of the samples converge at this point. 2. At this point, there are a few options: A. Perform Preamplification (next step) on both sample strips, but with different sets of target genes added. It will be important to use the same endogenous controls in all samples. This will become the standard option once the protocol has been established. B. Perform Preamplification (next step) on both samples strips. One can be used for Quantative- PCR analysis right away while the other can be frozen and stored. The 2 nd set of samples can be tested and compared to the first set to ensure that the act of freezing and thawing does not alter gene expression. This is important for reproducibility of technical replicates. C. Perform Preamplification (next step) on only 1 strip. The remaining strip will be frozen and remain as cdna. It can be preamplified in the event that you decide later on to compare the gene expression of another target gene in the same embryo. D. If you chose to do the RT- Control above, perform preamplification on both strips. This is an important control to confirm that the DNASE treatment was effective. Once again, we highly recommend this control. 5

PREAMPLIFICATION: NOTE: The reason why I include the 8X calculation is that when we collect a full set of embryos into an 8-well strip, we use them as biological replicates by adding the same condition to each embryo through the entire protocol. We don t expect there to be significant gene expression variation between samples, but by following these suggestion, we have both biological and technical replicates all done together. 1. Pool Primers for your targets of interest. Dilute in Tris-EDTA buffer ph 8.0 (TE Buffer). NOTE: According to Ambion protocol, Final Primer concentration must be 180nM (0.18 um). 2. As an example, we typically combine our qrt-pcr primers at a 2uM stock solution. 3. If we decide to add 4 primer pairs, with each primer pair at 0.18 um, then: Primer Pair Primer Stock (2uM) Volume for 8X Endogenous Control F+R 1 0.54 ul 4.32 ul Endogenous Control F+R 2 0.54 ul 4.32 ul Target Gene F+R 1 0.54 ul 4.32 ul Target Gene F+R 2 0.54 ul 4.32 ul Tris-EDTA ph 8.0 Buffer 3.84 ul 30.72 ul Total 6 ul per Reaction 48 ul Total 4. Prepare PreAmplification Reaction Mix: Component Volume per Reaction (1X) Volume Per Reaction (8X) Single Cell PreAmp Mix 5 ul 40 ul Pooled Primers at 0.18uM 6 ul 48 ul (from above) Total 11 ul 88 ul 5. Add 11 ul of PreAmplification Reaction Mix to each sample. 6. Perform PreAmplifcation Reaction in a ThermalCycler: Stage Step Temperature Time Holding Enzyme Activation 95 o C 10 min Cycling (14x) Denature 95 o C 15 sec Anneal/Extend 60 o C 4 min Holding Enzyme Deactivation 99 o C 10 min 7. Total Volume of PreAmplified cdna: 27 ul 8. Place tube on ICE or Store in -20 o C. 6

cdna Dilution for quantitative RT-PCR: NOTE: This is essentially identical to the Ambion Protocol. 1. Prepare a 1:20 dilution prior to use in qrt-pcr reactions using TE Buffer ph 8.0. 2. Example of Dilution amounts: Component Volumes for 1:20 Dilution PreAmp cdna 5uL 10uL 25uL 27uL TE Buffer ph 8.0 95uL 190uL 475uL 513uL Total 100uL 200uL 500uL 540uL 3. Our lab uses the SSO Advanced SYBR Green FAST qrt-pcr Protocol (BioRad 1725275) 4. Briefly, setup per reaction: Reagent Volume per Reaction (1X) Volume Per Reaction (8X) Final Concentration SSO 2X Master Mix 5 ul 40 ul 1X Primer (For. + Rev.) 2uM 1 ul 8 ul (from above) 0.2uM Template (1:20 Dilution) 4 ul Added Later <20ng Total 10 ul 80 ul - 5. The rest depends on the samples you collected and what instrument you have to run the qrt- PCR. 6. Some suggested plate setups are detailed below. 7

QUANTATATIVE RT-PCR PLATE SETUP: A Suggestion. NOTE: Feel free to setup the qrt-pcr reaction however you are used to! This is merely a suggestion based on our current set up and does not mean other methods will not work! 1. RT+ and RT-: Initial Tests to confirm absence of Genomic DNA contamination Embryo1 Embryo1 Embryo1 Embryo1 Embryo1 Embryo1 Embryo1 Embryo1 Embryo1 Embryo1 Embryo1 Embryo1 Embryo2 Embryo2 Embryo2 Embryo2 Embryo2 Embryo2 Embryo2 Embryo2 Embryo2 Embryo2 Embryo2 Embryo2 Embryo3 Embryo3 Embryo3 Embryo3 Embryo3 Embryo3 Embryo3 Embryo3 Embryo3 Embryo3 Embryo3 Embryo3 Embryo4 Embryo4 Embryo4 Embryo4 Embryo4 Embryo4 Embryo4 Embryo4 Embryo4 Embryo4 Embryo4 Embryo4 Embryo5 Embryo5 Embryo5 Embryo5 Embryo5 Embryo5 Embryo5 Embryo5 Embryo5 Embryo5 Embryo5 Embryo5 Embryo6 Embryo6 Embryo6 Embryo6 Embryo6 Embryo6 Embryo6 Embryo6 Embryo6 Embryo6 Embryo6 Embryo6 Embryo7 Embryo7 Embryo7 Embryo7 Embryo7 Embryo7 Embryo7 Embryo7 Embryo7 Embryo7 Embryo7 Embryo7 Embryo8 Embryo8 Embryo8 Embryo8 Embryo8 Embryo8 Embryo8 Embryo8 Embryo8 Embryo8 Embryo8 Embryo8 ------------------------------------------------- RT+ -------------------------------------------------- ----------------RT- -------------- Embryo1 Embryo1 Embryo1 Embryo1 Embryo1 Embryo1 Embryo1 Embryo1 Embryo1 Embryo1 Embryo1 Embryo1 Embryo2 Embryo2 Embryo2 Embryo2 Embryo2 Embryo2 Embryo2 Embryo2 Embryo2 Embryo2 Embryo2 Embryo2 Embryo3 Embryo3 Embryo3 Embryo3 Embryo3 Embryo3 Embryo3 Embryo3 Embryo3 Embryo3 Embryo3 Embryo3 Embryo4 Embryo4 Embryo4 Embryo4 Embryo4 Embryo4 Embryo4 Embryo4 Embryo4 Embryo4 Embryo4 Embryo4 Embryo5 Embryo5 Embryo5 Embryo5 Embryo5 Embryo5 Embryo5 Embryo5 Embryo5 Embryo5 Embryo5 Embryo5 Embryo6 Embryo6 Embryo6 Embryo6 Embryo6 Embryo6 Embryo6 Embryo6 Embryo6 Embryo6 Embryo6 Embryo6 Embryo7 Embryo7 Embryo7 Embryo7 Embryo7 Embryo7 Embryo7 Embryo7 Embryo7 Embryo7 Embryo7 Embryo7 Embryo8 Embryo8 Embryo8 Embryo8 Embryo8 Embryo8 Embryo8 Embryo8 Embryo8 Embryo8 Embryo8 Embryo8 ------------------------------------------------- RT+ -------------------------------------------------- ----------------RT- -------------- Endogenous Control 1 Endogenous Control 2 Target Gene 1 Target Gene 2 2. Typical Setup: Testing Target and Endogenous Control Expression as well as reproducibility after each stage of embryo is collected. Embryo1 Embryo1 Embryo1 Embryo1 Embryo1 Embryo1 Embryo1 Embryo1 Embryo1 Embryo1 Embryo1 Embryo1 Embryo2 Embryo2 Embryo2 Embryo2 Embryo2 Embryo2 Embryo2 Embryo2 Embryo2 Embryo2 Embryo2 Embryo2 Embryo3 Embryo3 Embryo3 Embryo3 Embryo3 Embryo3 Embryo3 Embryo3 Embryo3 Embryo3 Embryo3 Embryo3 Embryo4 Embryo4 Embryo4 Embryo4 Embryo4 Embryo4 Embryo4 Embryo4 Embryo4 Embryo4 Embryo4 Embryo4 Embryo5 Embryo5 Embryo5 Embryo5 Embryo5 Embryo5 Embryo5 Embryo5 Embryo5 Embryo5 Embryo5 Embryo5 Embryo6 Embryo6 Embryo6 Embryo6 Embryo6 Embryo6 Embryo6 Embryo6 Embryo6 Embryo6 Embryo6 Embryo6 Embryo7 Embryo7 Embryo7 Embryo7 Embryo7 Embryo7 Embryo7 Embryo7 Embryo7 Embryo7 Embryo7 Embryo7 Embryo8 Embryo8 Embryo8 Embryo8 Embryo8 Embryo8 Embryo8 Embryo8 Embryo8 Embryo8 Embryo8 Embryo8 Endogenous Control 1 Endogenous Control 2 Target Gene 1 Target Gene 2 8

3. Development Test: Once confident about RT-, technical and biological replicate reproducibility. If one Endogenous control outperforms the rest, just use one. Oocyte1 4C1 8C1 Blast1 Oocyte1 4C1 8C1 Blast1 Oocyte1 4C1 8C1 Blast1 Oocyte2 4C2 8C2 Blast2 Oocyte2 4C2 8C2 Blast2 Oocyte2 4C2 8C2 Blast2 Oocyte3 4C3 8C3 Blast3 Oocyte3 4C3 8C3 Blast3 Oocyte3 4C3 8C3 Blast3 Oocyte4 4C4 8C4 Blast4 Oocyte4 4C4 8C4 Blast4 Oocyte4 4C4 8C4 Blast4 Oocyte5 4C5 8C5 Blast5 Oocyte5 4C5 8C5 Blast5 Oocyte5 4C5 8C5 Blast5 Oocyte6 4C6 8C6 Blast6 Oocyte6 4C6 8C6 Blast6 Oocyte6 4C6 8C6 Blast6 Oocyte7 4C7 8C7 Blast7 Oocyte7 4C7 8C7 Blast7 Oocyte7 4C7 8C7 Blast7 Oocyte8 4C8 8C8 Blast8 Oocyte8 4C8 8C8 Blast8 Oocyte8 4C8 8C8 Blast8 Endogenous Control 1 Target Gene 1 Target Gene 2 9