Quantitative-PCR analysis for transcription inhibition of HSF-1 target gene Hsp90aa Measured in Cell-Based System Using RT-PCR - 2038-08_Inhibitor_Dose_CherryPick_Activity
A modified version of NIH-3T3 cells, are treated with potential inhibitors of HSF-1. Following a 90 minute incubation in the presence of the compounds, the cells are subjected to a 42C heat shock for 90 minutes, then return to 37C for an additional 1 hour. The cells are then lysed, and a reverse transcriptase reaction is performed to convert mRNA transcripts to cDNA. This cDNA is then used to perform quantitative-PCR (qPCR) analysis to measure the quantity of transcripts of the HSF-1 target gene Hsp90aa relative to a 'house-keeper' control gene, beta-actin. ..more
BioActive Compound: 1
Heat Shock Factor-1 (HSF-1), Heat Shock Response, NIH-3T3, qPCR, Hsp90aa
A modified version of NIH-3T3 cells, are treated with potential inhibitors of HSF-1. Following a 90 minute incubation in the presence of the compounds, the cells are subjected to a 42C heat shock for 90 minutes, then return to 37C for an additional 1 hour. The cells are then lysed, and a reverse transcriptase reaction is performed to convert mRNA transcripts to cDNA. This cDNA is then used to perform quantitative-PCR (qPCR) analysis to measure the quantity of transcripts of the HSF-1 target gene Hsp90aa relative to a 'house-keeper' control gene, beta-actin.
The compounds tested in this assay were previously identified as potential HSF-1 inhibitors in a HSF-1 regulated reporter-gene assay (AID 2098). This assay tests the ability of these compounds to impede the transcriptional activity of HSF-1 by assessing their ability to prevent transcription of the HSF-1 target gene Hsp90aa as determined by qPCR. Selective inhibitors of HSF-1 should reduce the transcript levels of Hsp90aa relative to a stably expressed 'house-keeping' gene beta-actin.
Quantitative-PCR analysis for transcription inhibition of HSF-1 target gene Hsp90aa:
The HGL cell line is modified version of NIH3T3 fibroblasts with an integrated GFP/Luciferase construct under the control of a heat shock response element.The HGL cell line was generously provided for this study by Dr. Luke Whitesell.
The HGL cell line is propagated in Opti-media (Invitrogen) supplemented with 5% heat inactivated fetal bovine serum (Invitrogen), 1% penicillin/streptomycin/glutamine at 37 degrees C in CO2 incubators (Thermo Scientific) with 5% CO2, 21% O2, and 95% humidity. For High-Throughput Screening assays, cells are grown in T225 flask (BD Falcon) or Hyperflasks (Corning), harvested at more than 80% confluence using Accumax (Innovative Cell Technologies). Cell number is counted using a Nexelcom Bioscience cell counter (Cellometer Auto M10) and viability is measured by mixing cells 1:1 with a 0.4% Trypan Blue solution (Sigma). Only cultures of >94% viability are utilized for experiments.
Compound Screening is carried out on the Broad Institute/Chemical Biology Platform Walk-Up Instruments:
HGL HSF-1 luc cells are harvested and resuspended in Opti-media with 2.5% Heat inactivated fetal bovine serum (Invitrogen), 1% penicillin/streptomycin/glutamine (Invitrogen). HGL cells (from an initial cell suspension of 375,000 cells/ml) are dispensed using a MultiDrop Combi/Standard tube dispensing cassette (Thermo Scientific) in white bottom 384 well assay plates (Corning) at a final density of 15,000 cells per well in final volume of 40 iL. The cells are kept in suspension using a magnetic bar and a stirrer during the dispensing.
The assay plate (cell plate) are placed in Thermo 3110 Incubator and incubated overnight at 37 degrees C, 5% CO2, 21% O2, and 95% humidity.
Compound pinning into assay plate:
Test compounds plates are pinned into duplicate assay plates using the 100nL pin array
90min incubation with the compounds, the plates are heat-shocked by being placed in a 42 degrees C incubator for 90min. Following heat-shock, the plates returned to 37 degrees C and further incubated for 1hr.
Cell to CT lysis:
The medium is aspirated and the cells are washed twice (100 uL with PBS) using the ELX405 Plate washer (Biotek). The assay plates are flipped upside down and centrifuged at 1000 rpm 2 minutes to remove the excess liquid.
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 are 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 (faced up)(1000 rpm, 2minutes) and 1 uL of stop solution is added with the Multidrop Combi-nl (Thermo Scientific). The assay plate are incubated for 2 minutes at room temperature and processed for reverse transcription.
Reverse transcription (RT)
(Ambion, Cell to CT RT Mix)
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 RT Master Mix is dispensed into each well of a RT assay plate (Axygen, PCR-384 RGD C). 2 uL of cell lysate are transferred into RT assay plate using a Vario transfer unit (CyBi Well).
The RT assay plates are placed in a DNA Engine Tetrad Thermocycler (MJ Research) and incubated at 37C for 1h, followed by a 1 minute incubation at 95C to inactivate the reverse transcriptase.
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
(murine Hsp90aa, Applied Biosystems,
cat# Mm00658568_gH, lot# 925665)
20X VIC Taqman probe/primers set 0.125 uL
(murine actin-beta, Applied Biosystems
cat# 4352341E, lot# 1009025)PCR H2O 1.25 uL
4 uL/well of PCR master mix is dispensed into a white Light Cycler-480 384 multi-well PCR plate (Roche) using the Multidrop Combi-nl (Thermo Scientific)
Then, 1 uL/well of RT DNA is transferred in the 4 uL/well PCR plate. The PCR plates are centrifuged (faced up) 2 minutes at 1000 rpm.
PCR is performed using a Light Cycler 480 II thermo-cycler (Roche) with the following conditions:
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. 25 degrees C infinity
PRESENCE OF CONTROLS: Neutral control wells (NC; n=36) and positive control wells (PC; n=36) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (2)(Max_Concentration).
The raw signals of the plate wells were normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral controls (NC) is set to a normalized activity value of 0.
A normalized activity value of 100 is defined as (2)(NC).
A normalized activity value of -50 is defined as (0.5)(NC).
Experimental wells values were scaled to this range.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
Categorized Comment - additional comments and annotations
Assay Format: Cell-based
Assay Cell Type: NIH 3T3
Assay Type: Functional
* Activity Concentration. ** Test Concentration.
Data Table (Concise)