REAGENT SETUP:
Primer Dilutions- Forward and reverse primers need to be ordered at a stock concentration of 400µM to obtain a forward reverse mix concentration of 200µM. Two additional working dilutions of 100 µM and 20 µM (forward and reverse primer mix).
a. 200 µM, 100 µM (forward and reverse primer mix) – Used in the Pre-amplification step (1.1 STEP 3)
b. 20 µM (forward and reverse primer mix) – Used for BioMark Chip loading (1.3 STEP 2)
c. 2 µM (forward and reverse primer mix)- Used for traditional qPCR (1.2)
Lysis Buffer- Mix the following components based on the number of samples in the experiment. Calculation shown for 1 and 96 samples with overage :
· For one sample: 0.5 μl of Lysis enhancer (CellDirect kit) + 5 μl of Resuspension buffer (CellDirect kit) for a total of 5.5 μl
· For 96 samples with overage 110 samples: 55 μl of Lysis enhancer (CellDirect kit) + 550 μl of Resuspension buffer (CellDirect kit) for a total of 605μl
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 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 two times the starting material as the previous point, therefore if the primer scales accurately there should be 1 Ct delta 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 for a single cell experiment 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.
RT Mix- To be prepared immediately prior to use in STEP 3. Mix the following components based on the number of samples in the experiment. Calculation shown for 96 samples with overage :
· For one sample: 0.30 μl 10X Superscript III Mix (VILO kit) + 0.2 μl of T4 Gene 32 Protein + 1 μl of DNA Suspension Buffer for a total of 1.5 μl
· For 96 samples with overage 110 samples: 33 μl 10X Superscript III Mix (VILO kit) + 22 μl of T4 Gene 32 Protein + 110 μl of DNA Suspension Buffer for a total of 165 μl
500nM Primer pool- 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. Below is the description for generating primer pools for 1-200 primers and 201-400 primers.
1. 1-200 primers – use primers at 100 μM concentration
· Combine 1 μl of each primer in a tube. Bring the volume up to 200 μl with DNA Suspension Buffer. For a primer pool of 96 primers; 96 μl of combined primer volume + 104 μl of DNA Suspension Buffer = 200 μl of primer pool at 500nM concentration.
2. 201-400 primers – use primers at 200 μM concentration
· Combine 1 μl of each primer in a tube. Bring the volume up to 400 μl with DNA Suspension Buffer. For a primer pool of 300 primers; 300 μl of combined primer volume + 100 μl of DNA Suspension Buffer = 400 μl of primer pool at 500nM concentration.
PreAmp Mix- To be prepared immediately prior to use in STEP 4. Mix the following components based on the number of samples in the experiment. Calculation shown for 96 samples with overage :
· For one sample: 10 μl of TaqMan PreAmp Master Mix of + 1.6 μl of 500 nM primer pool for a total of 11.6μl
· For 96 samples with overage 110 samples: 1100 μl of TaqMan PreAmp Master Mix + 176 μl of 500 nM primer pool for a total of
Exonuclease Mix To be prepared immediately prior to use in STEP 5. Mix the following components based on the number of samples in the experiment. Calculation shown for 96 samples with overage :
· For one sample: 0.8 μl of Exonuclease I reaction buffer 10X + 1.6 μl of Exonuclease I + 5.6 μl of DNA Suspension Buffer for a total of 8 μl
· For 96 samples with overage 110 samples: 88 μl of Exonuclease I reaction buffer 10X + 176 μl of Exonuclease I + 616 μl of DNA Suspension Buffer for a total of 880 μl
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.
EQUIPMENT SETUP:
1- Thermocyclers settings
a. VILO activation
65oC – 1 minute 30 seconds
4oC – ∞
b. RT
25oC – 5 minutes
50oC – 30 minutes
55oC – 25 minutes
60oC – 5 minutes
70oC – 10 minutes
4oC – ∞
c. Pre-amp 22 cycles
95oC – 10 minutes
22 cycles of:
96oC – 5 seconds
60oC – 4 minutes
4oC – ∞
d. Exonuclease
37oC – 30 minutes
80oC – 15 minutes
4oC – ∞
PROCEDURE
1.1 PROCEDURE FOR SAMPLE PRE-AMPLIFICATION:
Required for low input/single cell samples to selectively amplify cDNA to detectable levels for PCR.
STEP 1: Cell Lysis
o Add 5.5 μl lysis buffer (Reagent Preparation) to the capture surface on the LCM Cap
o Cover the cap with a 0.2 ml tube, ensure tight seal
o Incubate at 75 oC on a heat block (Cap surface in contact with the heating block) for 15 min
o Cool Cap and tube on ice for 5 minutes
o Spin down and transfer cell lysate to PCR tube or plate
Note: This step is for processing samples collected using LCM. For cell culture samples lyse cells in tubes instead of LCM cap.
STEP 2: Sample plate setup
Note: This step is performed on ice
a. Unknown samples: Transfer lysed cells from LCM caps to PCR plate. If using extracted RNA transfer 1 μl of RNA from sample (10-100pg) add 4.5 μl of lysis buffer (reagent preparation).
b. Positive control: Transfer 1 μl of RNA standard (Reagent preparation) to PCR plate and add 4.5 μl of lysis buffer.
c. Negative control: Add 1 μl of molecular grade water to a single well on the PCR plate and add 4.5 μl of lysis buffer.
STEP 3: Reverse Transcription
Note: This step is performed on ice
o 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).
o Spin down for 2 minutes at 2000 rpm and proceed to Thermocycler
o Thermocycler : VILO activation
o Add 1.5 μl of RT mix to each sample (reagent preparation)
o Spin down for 2 minutes at 2000 rpm and proceed to Thermocycler
o Thermocycler : RT
o Spin down for 2 minutes at 2000 rpm and place on ice
Note: This is an optional stopping point. Samples can be stored overnight at 4⁰C or stored at -20⁰C for a year.
STEP 4: Pre-amplification
Note: This step is performed on ice
o Add 11.6 μl of PreAmp Mix (reagent preparation) to each sample
o Spin down for 2 minutes at 2000 rpm and proceed to Thermocycler
o Thermocycler : PreAmp22
o Spin down for 2 minutes at 2000 rpm and place on ice
Note: This is an optional stopping point. Samples can be stored overnight at 4⁰C.
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.
STEP 5: Exonuclease treatment
o Add 8 μl of Exonuclease Mix (reagent preparation) to each sample
o Spin down for 2 minutes at 2000 rpm and proceed to Thermocycler
o Thermocycler : Exonuclease
o Spin down for 2 minutes at 2000 rpm and place on ice
o Add 54 μl of TE buffer to each sample
Note: This is an optional stopping point. Samples can be stored overnight at 4⁰C or stored at -20⁰C for a year.
1.2 PROCEDURE FOR TRADITIONAL QPCR:
o Samples can be measured using a traditional qPCR instrument using the following reaction mix for a 20 μl qPCR reaction:
2 μl of Pre-amplified and Exonuclease treated sample + 2 μl of Primer pair (2μM) + 10 μl of TaqMan Universal PCR Master Mix + 6 μl of Molecular grade water for a total of 20 μl.
o Run a standard 30-35 cycle PCR for gene expression.
1.3 PROCEDURE FOR RUNNING A BIOMARK CHIP:
STEP 1: Chip sample plate preparation
Note: Prepare Chip Sample plate on the day of Chip run
o Prepare Sample Mix
· For one Sample: 4 μl of 2X SsoFast EvaGreen Supermix with Low ROX + 0.4 μl of 20X DNA Binding Dye Sample Loading Reagent
· For a 48.48 IFC Biomark chip with overage for 55 samples: 220 μl of 2X SsoFast EvaGreen Supermix with Low ROX + 22 μl of 20X DNA Binding Dye Sample Loading Reagent
· 96.96 IFC Biomark chip with overage for 110 samples: 440 μl of 2X SsoFast EvaGreen Supermix with Low ROX + 44 μl of 20X DNA Binding Dye Sample Loading Reagent
o In a new PCR plate aliquot 4.4ul of the above Sample 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)
o Add 3.6 μl of Exonuclease treated sample to the plate with the above reagents
o Spin down for 2 minutes at 2000 rpm and place on ice
STEP 2: Chip assay plate preparation
Note: Chip assay plate can be prepared a day before chip run and stored at 4⁰C.
o In a new PCR plate aliquot 4 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 1 for the two types of IFC BioMark Chips.
o Add 4 μl of corresponding primer pairs at 20μM concentration to the wells.
o Spin down for 2 minutes at 2000 rpm and place on ice
Note: The final concentration of each primer pair is 5 μM in the inlet and 500 nM in the reaction chamber.
STEP 3: Priming and Loading BioMark Chip
o Inject control line fluid into each accumulator on the chip (see Figure 1).
o 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).
o When the Prime script has finished, press Eject to remove the primed chip from the IFC Controller.
o 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.
o Return the chip to the IFC Controller.
o 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.
o When the Load Mix script has finished, remove the loaded chip from the IFC Controller.
o Remove any dust particles or debris from the chip surface using scotch tape.
STEP 4: Data collection software for BioMark HD System
o Double-click the Data Collection Software icon on the desktop to launch the software.
o Click Start a New Run.
o Check the status bar to verify that the lamp and the camera are ready. Make sure both are green before proceeding.
o Remove and discard the blue protective film from the bottom of the chip. Place the chip into the reader. Click Load.
o Verify chip barcode and chip type. Click Next.
o Chip Run file: Select New. Browse to a file location for data storage. Click Next.
o Application, Reference, Probes:
o Select Application Type--Gene Expression for version 3.1.2 or higher software (for all earlier versions, contact Fluidigm Technical Support).
o Select Passive Reference: ROX.
o Select Probe--Single probe.
o Select probe type: EvaGreen. Click Next.
o 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