Ligation Mediated Amplification (LMA)
Transcript Capture (CAP)
Lysate and/or total RNA mixed with lysis buffer are transferred in a volume of 20ul to Turbocapture plates for mRNA capture. The plate wells are coated with oligo dT which allow for rapid mRNA capture. The plate is sealed, spun quickly to remove air bubbles and incubated at room temperature for 60 minutes. Once the mRNA is captured, the lysate is removed. This is done by unsealing the plate, inverting it face down onto a super rag, and then spinning in a centrifuge. The unbound material soaks into the super rag and is discarded. The plate is now ready for reverse transcription master mix.
Reverse Transcription (FIRST)
First strand cDNA is made using a 5ul per well master mix containing #units MMLV and #umol/l each dNTP. The plate is sealed, spun quickly to remove air bubbles, and incubated at 37°C for 90 minutes during which the mRNA serves as a template and the bound oligo dT as the primer. The newly formed cDNA template is now bound to the plate well. The plate is again washed by inverting into a super rag and spun in centrifuge to remove master mix. The plate is now ready for Probe Anneal master mix.
Probe Anneal (ANNL)
Upstream and downstream probes for each gene are annealed to the first strand cDNA. Each probe contains a universal primer site and a gene specific sequence. The upstream probe also contains a barcode for annealing to a Luminex bead in the detection process. A 5ul master mix is prepared using 10fmol??? each probe in a 1X taq ligase buffer. The plate is spun quickly to remove air bubbles and is then denatured at 95°C for 2 minutes and ramped down from 70°C to 40°C over 6 hours. Following the 6 hour incubation, the plate is again washed by inverting into a super rag and spun in centrifuge at 1000 rpm for 1 minute to remove master mix and all unbound probe. The plate is now ready for Ligation master mix.
Probe Ligation (LIG)
The probe pairs are ligated together forming a template for PCR. A 5 ul master mix is prepared containing ##units of taq ligase in 1X ligase buffer. The mix is added to the plate, which is then sealed and spun quickly to remove air bubbles. The plate is then incubated at 45°C for 60 minutes followed by a 65°C hold for 10 minutes. The plate is again washed by inverting into a super rag and spun in centrifuge at 1000 rpm for 1 minute to remove master mix. The plate is now ready for PCR master mix.
PCR Amplification (PCR)
The ligated probe template is PCR amplified using a set of universal primers. A 15 ul master mix containing 10 fmol??? T3 and T7 primers, ##fmol each dNTP, and ##units hot start taq in a 1X reaction buffer is prepared. The mix is added to the plate, which is then sealed and spun quickly to remove air bubbles. Then plate is then loaded in a Thermo Electron MBS 384 Satellite Thermal Cycler for PCR amplification. After an initial denature step at 95°C for 15 minutes, the. plates are cycled 29 times at 60°C. The primers anneal to the universal primer sites on the ligated probe pairs. The upstream primer contains a biotin needed for staining. The end product yields fixed length, barcoded, biotinylated amplicons. The amplicon is ready for Hybridization.
PCR amplicon is hybridized to barcoded Luminex beads. The barcode incorporated in the amplicon is complementary to the barcode on each Luminex bead so each gene anneals to a specific bead. A 5ul aliquot of PCR amplicon is transferred to a well containing 30 ul bead mix (about 350 beads/gene/well). The plate is sealed and incubated at 95C for 2 min, then incubated at 45C for 18 hours. Following incubation, the plate is spun at 3000 rpm for 1 minute to pellet beads. The plate is now ready for wash and stain.