High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

The overall goal of this procedure is to prepare and run 384 well assay plates for high throughput analysis of a highly homologous group of genes, such as the type one and three interferon subtypes. This is accomplished by first preparing standard serial dilution used for running the assay plates. Next, the primer probe and no template control working stock mixes used for making the plates are prepared.

Then the 384 well assay plates are prepared using an automated multi-channel pipetter and they are dried down for storage. Finally, the 384 well assay plates are run and analyzed. Ultimately the high throughput quantitative real-time RT PCR R assay is used to define interferon expression signatures in tissue culture models, studying pathogens or in clinical samples in the context of infectious or autoinflammatory diseases.

The main advantage of this technique over existing methods like quantitative R two PCR, using standard linear probes or cyberg green, is that molecular beacon and lock nucleic acid fluorescent probes can be used to discriminate single base pair differences between highly similar sequences After thawing, vortexing and briefly centrifuging a standard and salmon sperm or S-S-D-N-A. Prepare the SSD NA water mix for the 17 standard dilution sets by mixing 51 microliters of S-S-D-N-A with 20.3 milliliters of water. Dispense 190 microliters of the SS DNA water mix to the first tube of an eight tube PCR strip and 180 microliters to the next five tubes.

Perform a one to 20 dilution by transferring 10 microliters from the 50 Picomolar standard stock to the tube with 190 microliters of S-S-D-N-A water mix vortex and quickly centrifuge. The PCR strip perform a one to 10 dilution by transferring 20 microliters from the most recently diluted tube to the next tube in the series vortex and quickly centrifuge the PCR strip. Repeat the one to 10 dilution until the last tube in the series has received the standard.

After labeling up to 17 two milliliter tubes, use a 12.5 microliter electronic multi-channel pipette to add two microliters of S-S-D-N-A to every tube using primers and probes prepared according to the text protocol. Add the appropriate forward primer, reverse primer and probe to each primer. Probe set working stock tube to prepare the no template control or NTC working stock mixes.

Transfer 14.3 microliters of each primer probe set to the appropriate NTC working stock mix tube according to this table. Following the removal of an Eloqua of the primer probe sets required for the NTC working stock mixes at 1.5 milliliters of water to bring the final volume of each primer probe set working stock tube to 1.7 milliliters. To prepare a 96 well source plate of the primer probe sets and NTC mixes.

Place a new 96 well plate in a chilled 96 well cooling block and designate the wells with the correct primer probe set or NTC mix using a 250 microliter electronic multi-channel pipette. Add 66 microliters of water to every primer probe set. Well dispense 82.5 microliters of water to the four target 17 wells.

Dispense 27.5 microliters of water into every anti C mix. Well next, add 54 microliters of the correct primer probes at working stock to the designated primer probes at wells dispense. 67.5 microliters of the target 17 primer Probe working set stock to the designated target 17 primer probes at wells.

Add 22.5 microliters of the correct NTC working stock mix to the designated wells. Then use an adhesive plate seal to seal the 96 well plate and centrifuge for one minute at 700 Gs to ensure the contents are at the bottom of the wells with the 96 well adapter. Place the plate in the vortex mixer and mix at 1000 RP for one minute.

Then centrifuge the plate at 700 Gs for five minutes. To prepare the 384 well assay plates switch on the automated multichannel pipetter and double click on the software icon to open the protocol for making interferon assay plates for setting up the platform. Place a completely full pipette tip box in platform position one, the 96 well source plate in position four and a new 384 well plate in position six.

Begin the run by pressing the play button when the run is complete. Gently tap the 384 well assay plate on a flat surface to ensure fluids are at the bottom of the wells and apply an adhesive plate seal after spinning the plate at 700 Gs for one minute. Remove the adhesive plate seal.

Place the 384 well assay plate into the plate dryer and position the 384 well manifold directly above the wells. Once the 384 well assay plates contents are dry. Apply a new adhesive plate seal, rapid foil label and store in the dark at four degrees Celsius until use to load and run the assay plate.

Prepare two housekeeping gene or HKG well mixes by diluting two microliters of S-S-D-N-A in 84.9 microliters of water. Then add two microliters of the diluted SSD NA 11.8 microliters of water, 23 microliters of master mix and 9.2 microliters of the correct 20 XHKG primer Probe set to each labeled HKG tube, vortex, and briefly centrifuge after preparing the samples and positive control CDNA at 78.8 microliters of master mix and 54.8 microliters of water to each 24 microliter sample and positive control tube. To prepare the standards and NTC well mixes to be added to a dried 384 well assay plate add 165 microliters of water to 275 microliters of master mix in a 1.5 milliliter tube for the NTC well mix at 52.5 microliters of water to 52.5 microliters of master mix in a 1.5 milliliter tube.

Remove a previously prepared dried 384 well assay plate from four degrees Celsius and centrifuge at 700 GS for one minute. With the 30 microliter electronic multichannel pipette dispense 7.5 microliters of NTC. Well mix to each NTC.

Well dispense six microliters of the standards well mix and 1.5 microliters of the standard to each standard. Well then dispense 7.5 microliters of the sample to the designated wells with a 12.5 microliter electronic multichannel pipette. Dispense 2.5 microliters of the HKG primer Probe mixes to the designated wells after using optical adhesive film to seal the plate and centrifusion at 700 Gs for one minute vortex the sealed 384 well plate in the vortex mixer at 2, 600 RPM for two minutes and centrifuge at 700 cheese for five minutes.

Finally, place the sealed assay plate in the Q-R-T-P-C-R machine. Open the template for the assay layout and using the following reaction conditions. Begin the run, export and analyze the data according to the text protocol as seen in this figure, human type one and three interferon expression signatures were analyzed in peripheral blood mononuclear cells or p BMCs from six donors stimulated with toll-like receptor ligands.

The data are presented as radar charts in a log scale using two methods of analysis, including absolute CQ value normalized to the housekeeping gene and copy number of template per microgram of total RNA. The data show that human interferon expression signatures elicited by TLR agonists are ligand specific expression signatures of types one and three interferon in Resus Maccas. Were also TLR ligand specific in this experiment, PBMC from three donors were stimulated with three ligands for three hours.

Interferon subtype expression in unstimulated cells at baseline was low as shown here. A limited number of interferon alpha subtypes were expressed in response to LPS and poly ic. In contrast, interferon expression in response to imiquimod was high and the subtype expression was broad expression of interferon beta and interferon.

Lambda one was enhanced by all three TLR agonists At after watching this video, you should have a good understanding of how to prepare, run and analyze. 3D four well assay plates for high throughput analysis of a highly similar group of genes like the type one and three interferon subtypes.