May 04, 2020

Public workspaceSINGLE CELL HIGH-THROUGHPUT QRT-PCR PROTOCOL V.2

DOI
dx.doi.org/10.17504/protocols.io.bftdjni6
  • 1Thomas Jefferson University
  • SPARC
    Tech. support email: info@neuinfo.org
Sirisha Achanta
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.bftdjni6
Protocol Citation: Sirisha Achanta 2020. SINGLE CELL HIGH-THROUGHPUT QRT-PCR PROTOCOL. protocols.io https://dx.doi.org/10.17504/protocols.io.bftdjni6
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: April 30, 2020
Last Modified: May 04, 2020
Protocol Integer ID: 36421
Keywords: Biomark Fluidigm High Throughput Real Time PCR, Single cell, qRT-PCR, LCM, BioMark, qPCR
Abstract
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.
Materials
MATERIALS
ReagentT4 Gene 32 Protein - 100 ugNew England BiolabsCatalog #M0300S
ReagentExonuclease I (E.coli) - 3,000 unitsNew England BiolabsCatalog #M0293S
ReagentThermocycler
ReagentTaqMan™ PreAmp Master MixApplied Biosystems (ThermoFisher Scientific)Catalog #4391128
ReagentTaqMan Universal PCR Master MixLife TechnologiesCatalog #4304437
ReagentTE BufferInvitrogen - Thermo FisherCatalog #AM9861
ReagentCapSure HS CapsApplied Biosystems (ThermoFisher Scientific)Catalog #LCM0214
ReagentCellsDirect™ One-Step qRT-PCR KitInvitrogen - Thermo FisherCatalog #11753100
ReagentSuperScript™ VILO™ cDNA Synthesis KitInvitrogen - Thermo FisherCatalog #11754050
Reagent2X SsoFast EvaGreen Supermix with Low ROXBio-Rad LaboratoriesCatalog #1725211
Reagent20x DNA Binding Dye Sample Loading ReagentFluidigmCatalog #100-7609
Reagent2X Assay Loading ReagentFluidigmCatalog #100-7611
ReagentControl Line Fluid Kit—96.96FluidigmCatalog #89000021
ReagentControl Line Fluid Kit—48.48FluidigmCatalog #89000020
Reagent48.48 Dynamic Array™ IFC for Gene ExpressionFluidigmCatalog #BMK-M-48.48
Reagent96.96 Dynamic Array™ IFC for Gene ExpressionFluidigmCatalog #BMK-M-96.96
ReagentIFC Controller HXFluidigm
ReagentBioMark HD SystemFluidigm
Reagent Preparation
Reagent Preparation
2h
2h
Lysis Buffer: Combine Lysis enhancer and Resuspension buffer (CellsDirect Kit) as detailed in Table 1 (below)
Volume for one sample (μl) Volumes for 96 samples with overage ( for 110 samples) (μl)
Lysis enhancer (CellDirect kit) 0.5 55
Resuspension buffer (CellDirect kit) 5 550
Total 5.5 605
Table 1: Lysis Buffer Preparation
Note
Lysis Buffer can be stored at -20oC for upto a year

Primer Dilutions
Note
Forward and reverse primers need to be ordered at a stock concentration of 400µM to obtain a forward reverse mix concentration of 200µM.
Prepare primer dilutions:
  • Concentration100 micromolar (µM) , Concentration200 micromolar (µM) (forward and reverse primer mix) – Used in the pre-amplification step ( Step 9 ). Primer pool preparation see STEP 3
  • Concentration20 micromolar (µM) (forward and reverse primer mix) – Used for BioMark Chip loading (STEP 14)
  • Concentration2 micromolar (µM) used for traditional qPCR (STEP 17)

Primer Pool Preparation - Concentration500 nanomolar (nM) . 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)
Primer pool upto 200 primers Volume
Primer pairs (100 μM) 1 ul from each primer pair- X μl
DNA Suspension Buffer 200 – X μl
Total 200 μl
Table 2: 500 nM primer pool for 1-200 primers- Each primer is at a 500nM concentration.
Primer pool 201-400 primers Volume
Primer pairs (200 μM) 1 ul from each primer pair- X μl
DNA Suspension Buffer 400 – X μl
Total 400 μl
Table 3: 500 nM primer pool for 201-400 primers- Each primer is at a 500nM concentration.
Note
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.

Note
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.

Positive and Negative controls
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.
Sample Plate Setup
Sample Plate Setup
Cell Lysis
Note
This step is for processing samples collected using LCM. For cell culture samples lyse cells in tubes instead of LCM cap.
  • Add Amount5.5 µL lysis buffer (Reagent Preparation) to the capture surface on the LCM Cap
  • Cover the cap with a 0.2 ml tube, ensure tight seal
  • Incubate at Temperature75 °C on a heat block (Cap surface in contact with the heating block) for Duration00:15:00
  • Cool Cap and tube on iceTemperatureOn ice for Duration00:05:00
  • Spin down to collect lysate in the PCR tube and transfer cell lysate plate

Note
Refer to the LCM protocol for details on how to aquire samples from tissue sections.

Sample plate setup

Note
This step is performed on TemperatureOn ice


  • Unknown samples: Transfer cell lysate to PCR plate. If using extracted RNA transfer 1 μl of RNA from sample (10-100pg) add Amount4.5 µL of lysis buffer (Reagent Preparation).
  • Positive control: Transfer Amount1 µL of RNA standard (Reagent Preparation) to PCR plate and add Amount4.5 µL of lysis buffer.
  • Negative control: Add Amount1 µL of molecular grade water to a single well on the PCR plate and add Amount4.5 µL of lysis buffer.

Reverse Transcription
Reverse Transcription
Vilo activation

Note
This step is prepared TemperatureOn ice

  • Add 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 Duration00:02:00 at 2000 rpm and proceed to Thermocycler
  • Thermocycler : VILO activation
Temperature65 °C – Duration00:01:30
Hold at Temperature4 °C


RT Mix: To be prepared immediately prior to use in STEP 8. Mix the following reagents using the table below (Table 4)

Component Volume for one sample (μl) Volumes for 96 samples with overage ( for 110 samples) (μl)
10X Superscript III Mix (VILO kit) 0.30 33
T4 Gene 32 Protein 0.20 22
DNA Suspension Buffer 1 110
Total 1.5
Table 4: Reaction mix for Reverse Transcription

  • Add Amount1.5 µL of RT mix to each sample (Table 4)
  • Spin down forDuration00:02:00 at 2000 rpm and proceed to Thermocycler
  • Thermocycler : RT

Temperature25 °C forDuration00:05:00
Temperature50 °C forDuration00:30:00
Temperature55 °C forDuration00:25:00
Temperature60 °C forDuration00:05:00
Temperature70 °C forDuration00:10:00
Hold at Temperature4 °C
  • Spin down for Duration00:02:00 at 2000 rpm and place on ice

Note
This is an optional stopping point. Samples can be stored overnight at Temperature4 °C or stored at Temperature-20 °C for a year.




Pre-amplification
Pre-amplification
PreAmp Mix To be prepared immediately prior to use in STEP 10. Mix the following reagents using the table below (Table 5)
Component Volume for one sample (μl) Volumes for 96 samples with overage (for 110 samples) (μl)
TaqMan PreAmp Master Mix      10 1100
500 nM primer pool 1.6 176
Total 11.6
Table 5: Reactiom Mix for Pre-amplification
Pre-amplification

Note
This step is performed on TemperatureOn ice

  • Add Amount11.6 µL of PreAmp Mix to each sample
  • Spin down for 2 minutes at 2000 rpm and proceed to Thermocycler
  • Thermocycler : PreAmp22
Temperature95 °C for Duration00:10:00
22 cycles of:
Temperature96 °C for Duration00:00:05
Temperature60 °C for Duration00:04:00
Hold atTemperature4 °C
  • Spin down for Duration00:02:00 at 2000 rpm and place on ice
Note
This is an optional stopping point. Samples can be stored overnight at Temperature4 °C .

Note
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.



Exonuclease
Exonuclease
Exonuclease Mix: To be prepared immediately prior to use in STEP 12. Mix the following reagents using the table below (Table 6)

Component Volume for one sample (μl) Volumes for 96 samples with overage (for 110 samples) (μl)
Exonuclease I reaction buffer 10X 0.8 88
Exonuclease I  1.6 176
DNA Suspension Buffer 5.6 616
Total 8
Table 6: Reaction Mix for Exonuclease
Note
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 treatment:
  • Add Amount8 µL of Exonuclease Mix (reagent preparation) to each sample
  • Spin down for 2 minutes at 2000 rpm and proceed to Thermocycler
  • Thermocycler : Exonuclease

Temperature37 °C for Duration00:30:00
Temperature80 °C for Duration00:15:00
Hold atTemperature4 °C
  • Spin down for Duration00:02:00 at 2000 rpm andplace on ice
  • Add Amount54 µL of TE buffer to each sample


Note
This is an optional stopping point. Samples can be stored overnight at Temperature4 °C or stored atTemperature-20 °C for a year.






High-throughput qRT-PCR : BioMark Chip Run
High-throughput qRT-PCR : BioMark Chip Run
Chip Sample plate preparation

Note
Prepare Chip Sample plate on the day of Chip run

  • Combine 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) with overageVolume for 96.96 IFC (μl) with overage
2X SsoFast EvaGreen Supermix with Low ROX 4 220 440
20X DNA Binding Dye Sample Loading Reagent 0.4 22 44
Total 4.4
Table 7: Chip Sample Plate Reagent mix

  • In a new PCR plate aliquot 4.4ul of the above mix into sample wells in the following order
  • 96.96 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 Amount3.6 µL of Exonuclease treated sample to the plate with the above reagents
  • Spin down for Duration00:02:00 at 2000 rpm and place TemperatureOn ice
Chip Assay plate
Note
Chip assay plate can be prepared a day before chip run and stored at 4⁰C.
  • In a new PCR plate aliquot Amount4 µL 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 Amount4 µL of corresponding primer pairs at 20μM concentration to the wells
  • Spin down for Duration00:02:00 at 2000 rpm and place TemperatureOn ice

Note
The final concentration of each primer pair is 5 μM in the inlet and 500 nM in the reaction chamber

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.
Download Single Cell HT-qRT-PCR Figure 1.pdfSingle Cell HT-qRT-PCR Figure 1.pdf

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

Traditional qPCR Setup
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)
Component Volume (μl)
Pre-amplified and Exonuclease treated sample 2
Primer pair (2μM) 2
TaqMan Universal PCR Master Mix 10
Molecular grade water 6
Total 20
Table 8: PCR reaction components and volumes
Run a standard 30-35 cycle PCR for gene expression.