A novel, non-radioactive eukaryotic in vitro transcription assay for sensitive quantification of RNA polymerase II activity

Background Many studies of the eukaryotic transcription mechanism and its regulation rely on in vitro assays. Conventional RNA polymerase II transcription assays are based on radioactive labelling of the newly synthesized RNA. Due to the inefficient in vitro transcription, the detection of the RNA involving purification and gel electrophoresis is laborious and not always quantitative. Results Herein, we describe a new, non-radioactive, robust and reproducible eukaryotic in vitro transcription assay that has been established in our laboratory. Upon transcription, the newly synthesized RNA is directly detected and quantified using the QuantiGene assay. Alternatively, the RNA can be purified and a primer extension followed by PCR detection or qPCR quantification can be performed. When applied to assess the activity of RNA polymerase II inhibitors, this new method allowed an accurate estimation of their relative potency. Conclusions Our novel assay provides a non-radioactive alternative to a standard in vitro transcription assay that allows for sensitive detection and precise quantification of the newly transcribed, unlabelled RNA and is particularly useful for quantification of strong transcriptional inhibitors like α-amanitin. Moreover, the method can be easily adapted to quantify the reaction yield and the transcription efficiency of other eukaryotic in vitro systems, thus providing a complementary tool for the field of transcriptional research.

 Transfer 500µl beads (10mg/ml) to a fresh tube, remove the supernatant using a magnet, resuspend the beads into 1000µl Solution A (0.1M NaOH, 0.05M KCl) and incubate the beads 2min in solution A. Repeat this step once.
 Wash the beads once using 1000µl Solution B (0.1M KCl)  Wash the beads three times using 1000µl 1x B&W buffer (5mM Tris-HCl pH 7.5; 0.5mM EDTA; 1M NaCl) ii. DNA binding:  Place the tube into a magnet and remove the supernatant. Resuspend the beads into 900µl 1x B&W buffer and add 100µl of the Btn-HN-DNA (250µg/ml)  Incubate 15min at room temperature. Mix by gentle vortexing.  Place the tube into a magnet and transfer the supernatant to a fresh tube. Check DNA concentration and calculate the bead DNA loading yield. Typically a yield of 80% will be achieved.
 Wash the DNA-beads three times using 1000µl 1x B&W buffer.  Resuspend the DNA-beads into 1000µl 1x B&W buffer and store at 4°C. This bead suspension at 5mg/ml beads will typically contain 20µg/ml DNA.

Transcription reaction setup
a. Planning for the reaction: i. Prepare additional transcription reaction buffer (20mM HEPES pH 7.9, 100mM KCl, 0.2mM EDTA, 0.5mM DTT, 20% glycerol) by diluting RNase-free solutions with water. Store the buffer in frozen aliquots. ii.
Determine the volume of the 'HeLaScribe® Nuclear Extract' from the 'HeLaScribe® Nuclear Extract in vitro Transcription System' that corresponds to 8 activity units  x. Calculate the 'buffer volume' corresponding to 11μl-x. b. Before reaction setup: i. Calculate the DNA amount necessary to set up the desired number of reactions considering 150ng DNA per 25µl reaction. Resuspend the DNA-loaded beads and remove an aliquot that contains the desired amount of DNA. Typically, this will be 7.5µl DNA-bead suspension per reaction. Wash this aliquot two times in the double volume of transcription buffer using a magnet and resuspend for a third wash. Keep on ice until the inhibitor dilution series is prepared. ii.
Prepare the transcription inhibitor dilutions as 5-fold of the desired final concentration. For example, prepare a 5µM amanitin solution in order to test α-amanitin at 1µM. c. Reaction setup: i.
Resuspend the DNA-beads in transcription buffer in a volume corresponding to the 'buffer volume', i.e. 11µl-x per reaction. ii.
Gently thaw a tube of extract on ice and mix by gentle pipetting. Transfer the required extract at x µl per reaction into the tube containing the beads. iii.
Aliquot 19µl per reaction into the reaction tubes. Add 5µl of inhibitor dilution for the inhibited reactions or H 2 O for the positive and negative reactions. Mix by gentle vortexing and incubate for 20min at room temperature. v.
Add 1µl of the NTP mix to each tube except for the negative control. Mix by gentle vortexing and incubate for 30min at 30°C. During the incubation period, gentle vortex the reaction tubes 1-2 times to resuspend the beads. vi.
Centrifuge the reaction tubes for 10min at 4°C and maximal speed. Continue with step 3A or 3B.
3A. RNA detection by primer extension and qPCR quantitation a. RNA purification using RNeasy Mini kit i. Aliquot 400µl RLT buffer into fresh tubes. ii.
After transcription and centrifugation, transfer 20µl of the bead supernatant to the RLT buffer. Mix by pipetting. Store the samples at -80°C until proceeding. iii.
Transfer the sample to an RNeasy column. Centrifuge or apply vacuum. Discard the flow-through. v.
Apply 350µl buffer RW1 to the column. Centrifuge or apply vacuum. Discard the flow-through. vi.
Prepare a DNase digestion mix using the Epicentre DNase Zero kit or alternative. Per column 160µl digestion mix containing 50 DNase units/ml will be needed. vii.
Apply 160µl DNase digestion mix to the column. Incubate 15min at room temperature. viii.
Apply 350µl buffer RW1 to the column. Centrifuge or apply vacuum. Discard the flow-through. ix.
Apply 500µl buffer RPE supplemented with EtOH to the column. Centrifuge or apply vacuum. Discard the flow-through. x.
Apply 500µl 80%EtOH to the column. Centrifuge or apply vacuum. Discard the flow-through. xi.
Transfer the column to a dry collection tube. Centrifuge at maximal speed for 1min to completely dry the membrane. xii.
Place the column into a fresh elution tube. iii.
Distribute the working reagent mix as 95µl aliquots into the capture plate. iv.
Add 5µl diluted reaction mix to each well containing working reagent. Avoid introducing air bubbles. If desired, an RNA standard curve can be also prepared to be used for signal quantification. v.
Centrifuge the plate for 20s at 240 x g and room temperature. vi.
Seal the plate and incubate overnight at 55°C. vii.
Next day proceed with signal amplification and detection, as described in the Quantigene manual.