qPCR LDLR Timecourse Measured in Cell-Based System Using RT-PCR - 2143-24_Activator_Dose_CherryPick_Activity
- Deposit:2014-06-10
- Hold-Until:2015-06-10
- Modify:2014-06-10
Keywords: Trib1, LDLR, LDL-Cholesterol
Assay Overview: LDL uptake by liver cells is the primary mechanism for clearing LDL from the circulation. Compounds that increase the expression of the receptor for LDL (LDLR) are of interest. It needs to be determined if compounds that induce Trib1 gene expression also induce LDLR gene expression. HepG2 cells, a hepatocarcinoma cell line, were plated in 384 well plates and treated with Trib1 inducing compounds for 24 hr after which time the cells were lysed and cDNA was generated. A qPCR reaction determining LDLR mRNA expression was performed using GAPDH as an endogenous control gene to identify compounds that induce LDLR expression during a 0.5-28 hr timecourse.
Expected Outcome: Increase in LDLR expression
Day 1 (Cell Plating)
2000 HepG2 cells are plated in each well of a Corning White, 384-well, tissue culture plate in 40iL of DMEM media using a standard Combi liquid dispenser (Thermo). The plated cells are then incubated overnight in a 3110 tissue culture incubator (Thermo) at 37 degrees C and 5% CO2.
Day 2 (Compound Pinning)
The following day, 100nL of compound is added to each well using the Cy-Bi Well pin transfer array. After addition of compound, the plates are returned to the tissue culture incubator and are incubated for 0.5, 2, 4, 8, 24 or 28 hr at which time cDNA was made.
Day 3 (cDNA Synthesis)
1- Cells to CT lysis
The medium is aspirated and the cells are washed twice with 100 uL with PBS using the ELX405 Plate washer (Biotek). The assay plates are flipped upside down and centrifuged at 1000 rpm, 0.5 minutes to remove the remaining PBS. Ten (10) uL of Lysis solution with DNase I (Ambion, from Cell to CT Lysis Mix) is added to each well using the MultiDrop Combi/Standard tube dispensing cassette (Thermo Scientific). Each assay plate is then vortexed for 2 minutes and incubated for an additional 5 minutes at room temperature. At the end of the incubation, the assay plates are centrifuged (face-up)(1000 rpm, 0.5 minutes) and 1 uL of stop solution is added with the Multidrop Combi-nl (Thermo Scientific). The assay plates are incubated for 2 minutes at room temperature and processed for reverse transcription.
2- Reverse transcription (RT) (Ambion, Cell to CT RT #4402958)
TABLE 1. 8 uL RT Master Mix/10 uL total reaction volume
Component Amount per reaction
2X RT Buffer 5 uL
20X RT Enzyme Mix 0.5 uL
Nuclease-Free Water 2.5 uL
8 uL of the RT Master Mix is dispensed into each well of a RT assay plate (Axygen, PCR-384 RGD C). Two (2) uL of the cell lysate is transferred into the RT assay plate using the Vario transfer unit (CyBi Well). The RT assay plates are incubated at 37C for 1h and the reverse transcriptase is inactivated by incubating the plates for 5 minutes at 95C. cDNA is used immeadiately or stored at -80 degrees C until ready for qPCR analysis.
Day 3 or 4 (qPCR)
Table 2.
4 uL PCR Master Mix/5uL total volume of reaction
Component Amount per reaction
2X Roche Master Mix (Probes Master) 2.5 uL
20X FAM Taqman probe/primers set 0.125 uL
(human LDLR, IDT, Hs.PT.49a.20193252)
20X VIC Taqman probe/primers set 0.125 uL
(human GAPDH, Applied Biosystems 4326317E VIC-MGB)PCR H2O 1.25 uL
Four (4) uL/well of the PCR master mix is dispensed into the qPCR plate (Roche Light Cycler 480 MultiWell Plate 384, Cat# 04 729 749 001) using the Multidrop Combi-nl (Thermo Scientific).
One (1) uL/well of cDNA is transferred to the 4 uL/well qPCR plate. The qPCR plates are centrifuged for 0.5 minutes at 1000 rpm.
qPCR is performed using the Roche Light Cycler 480 II using the following protocol:
Step Temperature Time
1. 95 degrees C 10 minutes
2. 95 degrees C 10 seconds
3. 60 degrees C 30 seconds
Step 2 and 3 (55 cycles)
4. 40 degrees C 30 seconds