TITLE: SINGLE CELL HIGH-THROUGHPUT QRT-PCR PROTOCOL
AUTHORS: Sirisha Achanta

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Single cell high-throughput qRT-PCR protocol combines high sensitivity technique of single cell qPCR with high-throughput qPCR technology that can generate data from 96 samples and 96 genes in a single experiment. It can be adapted for various sample types- cell culture, tissue samples and extracted RNA (10pg) and measured on traditional qPCR and high-throughput qPCR platforms. The workflow is comprised of four steps – cell lysis, reverse transcription, pre-amplification and qPCR. Key features of this protocol are; processing low input samples directly to reverse transcription without RNA extraction which minimizes sample loss, pre-amplification enables amplification of cDNA from single cells to detectable levels for qPCR and measuring up to 400 genes from a single cell sample/10pg of RNA (starting material). Robust reproducible and versatile this protocol can be adapted to several upstream and downstream techniques.</div></div>

[STEPS]
?. [Reagent Preparation]
Lysis Buffer:  Combine Lysis enhancer and Resuspension buffer (CellsDirect Kit) as detailed in Table 1 (below)  ABC1       Volume
  for one sample (μl)    Volumes
  for 96 samples with overage ( for 110 samples) (μl)  2  Lysis enhancer
  (CellDirect kit)    0.5     55  3  Resuspension
  buffer (CellDirect kit)    5     550  4  Total    5.5    605
ABC1       Volume
  for one sample (μl)    Volumes
  for 96 samples with overage ( for 110 samples) (μl)  2  Lysis enhancer
  (CellDirect kit)    0.5     55  3  Resuspension
  buffer (CellDirect kit)    5     550  4  Total    5.5    605
Lysis Buffer can be stored at -20oC for upto a year
?. [Positive and Negative controls]
RNA Dilution series- This is used as a positive control for the experiment and must contain known amounts of RNA obtained from whole tissue and include all the conditions tested in the experiment. This is to ensure that the positive control has expression of all the genes measured. Dilution series is also used as a metric to test primer efficiency. For example in a 2X dilution series each point contains twice the starting material as the previous point, therefore if the primer scales accurately there should be 1 Ct difference between 2 dilution points.Prepare 2X or 3X RNA dilution series for 6-8 dilution points. The mean sample input should correspond to the middle points in the dilution series. For example, dilution series for an experiment with single cell samples (we consider a cell to contain approximately 10pg of RNA):2X dilution series – 2pg/ μl, 4pg/ μl, 8pg/ μl, 16pg/ μl, 32pg/ μl and 64pg/ μl.
?. [Reverse Transcription]
RT Mix: To be prepared immediately prior to use in STEP 8. Mix the following reagents using the table below (Table 4)   ABC1Component  Volume for one sample (μl)    Volumes for 96 samples with overage ( for
  110 samples) (μl)  2  10X Superscript III Mix (VILO kit)    0.30     33  3  T4 Gene 32 Protein    0.20     22     4  DNA Suspension Buffer    1    110  5  Total    1.5          Add  of RT mix to each sample (Table 4)Spin down for at 2000 rpm and proceed to Thermocyclerforforforforfor        Hold at Spin down for   at 2000 rpm and place on ice
ABC1Component  Volume for one sample (μl)    Volumes for 96 samples with overage ( for
  110 samples) (μl)  2  10X Superscript III Mix (VILO kit)    0.30     33  3  T4 Gene 32 Protein    0.20     22     4  DNA Suspension Buffer    1    110  5  Total    1.5
1.5 µl
25 °C
50 °C
55 °C
60 °C
70 °C
4 °C
This is an optional stopping point. Samples can be stored overnight at    or stored at   for a year.
?. [Pre-amplification]
PreAmp Mix To be prepared immediately prior to use in STEP 10. Mix the following reagents using the table below (Table 5)  ABC1Component  Volume for one sample (μl)    Volumes for 96 samples with overage (for
  110 samples) (μl)  2  TaqMan PreAmp Master Mix&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;     10    1100  3  500 nM primer pool    1.6     176  4  Total    11.6
ABC1Component  Volume for one sample (μl)    Volumes for 96 samples with overage (for
  110 samples) (μl)  2  TaqMan PreAmp Master Mix&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;     10    1100  3  500 nM primer pool    1.6     176  4  Total    11.6
?. [Exonuclease]
Exonuclease Mix: To be prepared immediately prior to use in STEP 12. Mix the following reagents using the table below (Table 6)  ABC1Component  Volume for one sample (μl)    Volumes for 96 samples with overage (for
  110 samples) (μl)  2  Exonuclease I reaction buffer 10X    0.8    88  3  Exonuclease I&nbsp;     1.6     176  4  DNA Suspension Buffer    5.6    616  5  Total    8
ABC1Component  Volume for one sample (μl)    Volumes for 96 samples with overage (for
  110 samples) (μl)  2  Exonuclease I reaction buffer 10X    0.8    88  3  Exonuclease I&nbsp;     1.6     176  4  DNA Suspension Buffer    5.6    616  5  Total    8
Catalyzes the removal of nucleotides from linear single-stranded DNA in the 3' to 5' direction. It is used for sample cleanup post pre-amplification to remove unincorporated primers and other single stranded cDNA.
?. [Exonuclease]
Exonuclease treatment: Add   of Exonuclease Mix (reagent preparation) to each sampleSpin down for 2 minutes at 2000 rpm and proceed to ThermocyclerThermocycler : Exonucleasefor for               Hold atSpin down for   at 2000 rpm andplace on iceAdd   of TE buffer to each sample
8 µl
37 °C
80 °C
4 °C
54 µl
This is an optional stopping point. Samples can be stored overnight at   or stored at   for a year.
?. [Reagent Preparation]
Primer DilutionsPrepare primer dilutions: ,   (forward and reverse primer mix) – Used in the pre-amplification step ( Step 9 ). Primer pool preparation see STEP 3  (forward and reverse primer mix) – Used for BioMark Chip loading (STEP 14)  used for traditional qPCR (STEP 17)
Forward and reverse primers need to be ordered at a stock concentration of 400µM to obtain a forward reverse mix concentration of 200µM.
?. [Reagent Preparation]
Primer Pool Preparation -  . For primer pool with fewer than 200 primers, see Table 2. For primer pool greater than 200 primers and less than 400 see Table 3 (below)  AB1  Primer pool upto 200 primers  Volume2  Primer pairs (100 μM)    1 ul from each primer pair- X μl  3  DNA Suspension Buffer    200 – X μl  4  Total    200 μl    AB1  Primer pool 201-400 primers  Volume2  Primer pairs (200 μM)    1 ul from each primer pair- X μl  3  DNA Suspension Buffer    400 – X μl  4  Total    400 μl
AB1  Primer pool upto 200 primers  Volume2  Primer pairs (100 μM)    1 ul from each primer pair- X μl  3  DNA Suspension Buffer    200 – X μl  4  Total    200 μl
AB1  Primer pool 201-400 primers  Volume2  Primer pairs (200 μM)    1 ul from each primer pair- X μl  3  DNA Suspension Buffer    400 – X μl  4  Total    400 μl
Primer pool can be generated and used for multiple experiments. Store at -20⁰C for a year. With 200 μM stock primers up to 400 primer pairs can be included. For generating primer pools with greater than 400 primer pairs, primers will have to be ordered at a higher concentration.
For generating primer pools with greater than 400 primer pairs, primers will have to be ordered at a higher concentration. We have tested up to 400 primers in the primer pool.
?. [Sample Plate Setup]
Cell LysisAdd  lysis buffer (Reagent Preparation) to the capture surface on the LCM CapCover the cap with a 0.2 ml tube, ensure tight sealIncubate at    on a heat block (Cap surface in contact with the heating block) for   Cool Cap and tube on ice   for    Spin down to collect lysate in the PCR tube and transfer cell lysate plateIncubate at   on a heat block (Cap surface in contact with the heating block) for Cool Cap and tube on ice  for
This step is for processing samples collected using LCM. For cell culture samples lyse cells in tubes instead of LCM cap.
5.5 µl
75 °C
on ice
Refer to the LCM protocol for details on how to aquire samples from tissue sections.
?. [Sample Plate Setup]
Sample plate setupUnknown samples: Transfer cell lysate to PCR plate. If using extracted RNA transfer 1 μl of RNA from sample (10-100pg) add   of lysis buffer (Reagent Preparation).Positive control: Transfer   of RNA standard (Reagent Preparation) to PCR plate and add   of lysis buffer.Negative control: Add   of molecular grade water to a single well on the PCR plate and add   of lysis buffer.
This step is performed on
4.5 µl
1 µl
4.5 µl
1 µl
4.5 µl
?. [Reverse Transcription]
Vilo activationAdd 1.8 μl of 5X VILO Reaction Mix ( Component of Superscript VILO cDNA synthesis kit) to each sample (unknown sample, positive control and negative control). Spin down for    at 2000 rpm and proceed to ThermocyclerThermocycler : VILO activationSpin down for   at 2000 rpm and proceed to Thermocycler  –             Hold at
This step is prepared
65 °C
4 °C
?. [Pre-amplification]
Pre-amplificationAdd   of PreAmp Mix  to each sampleSpin down for 2 minutes at 2000 rpm and proceed to ThermocyclerThermocycler : PreAmp22for             22 cycles of:for for         Hold atSpin down for   at 2000 rpm and place on ice
This step is performed on
11.6 µl
95 °C
96 °C
60 °C
4 °C
This is an optional stopping point. Samples can be stored overnight at   .
We consider 22 cycles of pre-amp optimal for single cells collected using LCM. We do not recommended to pre-amplification of samples over 22 cycles due to observed increase in production of non-specific PCR fragments and primer-dimers.
?. [High-throughput qRT-PCR : BioMark Chip Run]
Chip Assay plateIn a new PCR plate aliquot   2X Assay Loading Reagent into 48 or 96 wells for a 48.48 or 96.96 IFC Biomark chip using the corresponding layout as described in step 13 for the two types of IFC BioMark Chips.Add   of corresponding primer pairs at 20μM concentration to the wellsSpin down for   at 2000 rpm and place
Chip assay plate can be prepared a day before chip run and stored at 4⁰C.
4 µl
4 µl
on ice
The final concentration of each primer pair is 5 μM in the inlet and 500 nM in the reaction chamber
?. [High-throughput qRT-PCR : BioMark Chip Run]
Chip Sample plate preparationCombine 2X SsoFast EvaGreen Supermix with Low ROX and 20X DNA Binding Dye Sample Loading Reagent for a 48.48 or 96.96 IFC Biomark chip using the table below (Table 7):      Component      Volume per inlet with overage (μl)      Volume for 48.48 IFC (μl)      Volume for 96.96 IFC (μl)        2X SsoFast EvaGreen Supermix  with Low ROX      4      220      440        20X DNA Binding Dye Sample  Loading Reagent      0.4      22      44        Total      4.4                 In a new PCR plate aliquot 4.4ul of the above mix into sample wells in the following order96.96 IFC – fill wells A1 to H12 (all the wells)48.48 IFC – fill wells 1 to 6 in each row (A1-A6, B1-B6….H1-H6)   ABCD1  Component    Volume per inlet with overage (μl)  Volume for 48.48 IFC (μl)  with overageVolume for 96.96 IFC (μl)  with overage2  2X SsoFast EvaGreen Supermix  with Low ROX    4    220    440  3  20X DNA Binding Dye Sample  Loading Reagent    0.4    22    44  4  Total    4.4            In a new PCR plate aliquot 4.4ul of the above mix into sample wells in the following order96.96 IFC – fill wells A1 to H12 (all the wells)48.48 IFC – fill wells 1 to 6 in each row (A1-A6, B1-B6….H1-H6) Add   of Exonuclease treated sample to the plate with the above reagentsSpin down for   at 2000 rpm and place
Prepare Chip Sample plate on the day of Chip run
ABCD1  Component    Volume per inlet with overage (μl)  Volume for 48.48 IFC (μl)  with overageVolume for 96.96 IFC (μl)  with overage2  2X SsoFast EvaGreen Supermix  with Low ROX    4    220    440  3  20X DNA Binding Dye Sample  Loading Reagent    0.4    22    44  4  Total    4.4
3.6 µl
on ice
?. [High-throughput qRT-PCR : BioMark Chip Run]
Priming and loading BioMark Chip:Inject control line fluid into each accumulator on the chip (see Figure 1).Place the chip into the IFC Controller MX (for the 48.48 Chip) or the IFC Controller HX (for the 96.96 Chip), then run the Prime (113x) script (for the 48.48 Chip) or the Prime (136x) script (for the 96.96 Chip).When the Prime script has finished, press Eject to remove the primed chip from the IFC Controller.Pipette 5 μL of each assay and 5 μL of each sample into their respective inlets on the chip (see Figure 1). Using an 8 channel pipette load column 1 (A1, B1..H1) in the alternate wells starting with the top left well. Similarly load the next 5 columns for 96.96 chip and next 2 for 48.48 chip. For the seventh( 96.96) or the fourth (48.48 chip) column load starting with second from the top on the chip.Return the chip to the IFC Controller.Using the IFC Controller software, run the Load Mix (113x) script (for the 48.48 Chip) or Load Mix (136x) script (for the 96.96 Chip) to load the samples and assays into the chip chambers.When the Load Mix script has finished, remove the loaded chip from the IFC Controller.Remove any dust particles or debris from the chip surface using scotch tape.
?. [High-throughput qRT-PCR : BioMark Chip Run]
Data Collection software settings:Double-click the Data Collection Software icon on the desktop to launch the software.Click Start a New Run.Check the status bar to verify that the lamp and the camera are ready. Make sure both are green before proceeding. Remove and discard the blue protective film from the bottom of the chip. Place the chip into the reader. Click Load.Verify chip barcode and chip type. Click Next.Chip Run file: Select New. Browse to a file location for data storage. Click Next.Application, Reference, Probes: Select Application Type--Gene Expression for version 3.1.2 or higher software (for all earlier versions, contact Fluidigm Technical Support).Select Passive Reference: ROX.Select Probe--Single probe.Select probe type: EvaGreen. Click Next.Click Browse to find the thermal cycling protocol file.        For BioMark HD: GE Fast 48x48 PCR+Melt v2.pcl GE Fast 96x96 PCR+Melt v2.pcl        For BioMark HD: GE Fast 48x48 PCR+Melt v2.pcl GE Fast 96x96 PCR+Melt v2.pcl
?. [Traditional qPCR Setup]
Samples can be measured using a traditional qPCR instrument using the following reaction mix for a 20 μl qPCR reaction see Table 8 (below)  AB1  Component    Volume (μl)  2  Pre-amplified and Exonuclease treated sample    2  3  Primer pair (2μM)    2  4TaqMan Universal PCR Master Mix  10  5  Molecular grade water    6  6  Total    20   Run a standard 30-35 cycle PCR for gene expression.
AB1  Component    Volume (μl)  2  Pre-amplified and Exonuclease treated sample    2  3  Primer pair (2μM)    2  4TaqMan Universal PCR Master Mix  10  5  Molecular grade water    6  6  Total    20