Anscribed and PCR amplified from 10 ng of total RNA with gene specific primers and a florescent reporter probe that hybridises to the gene in between the two primers. The sequences of the primers were:following their specifications. About 3 of total RNA was mixed with 50 ng of random hexamers, and 1 mM dNTPs in 10 reaction volume and incubated at 65 for 5′ and left on ice for 1′. A cocktail mix of RT buffer, MgCl2 and DTT was added to a final concentration of 1X, 5 mM and 10 mM respectively along with 40 Units of RNase OUT Ribonuclease Inhibitor and incubated at 25 for 2′. 50 Units of Procyanidin B1 manufacturer SuperScript II Reverse Transcriptase enzyme was added and incubated at 25 for 10′ and 42 for 50′. The reaction was terminated by heating at 70 for 15′ and chilled on ice. 2 Units of RNase H was added and incubated at 37 for 20′ to remove the RNA from cDNA: RNA hybrids to increase the sensitivity of PCR from cDNA. Mpl was amplified using the Expand High Fidelity PCR kit from Roche, Indianapolis, IN, with gene specific primers containing EcoR 1 (FP) and Xho 1 (RP) Restriction Enzyme sites to enable directional cloning. The Restriction Enzyme sites are indicated in lower case in the Primer sequences given below. FP 5′-CGgaattcGAAGGGAGGATGGGCTAAGGC-3′ RP 5′-CCGctcgagAGTTTAGCTCTGTCCAGGGAA-3′ The Products were purified using the Wizard PCR Preps DNA Purification System from Promega, Madison, WI, and digested with the Restriction Enzymes, EcoR 1 and Xho 1. The digested products were then cloned into Blue Script vector. The recombinants were checked by restriction analysis and the selected positive clones were sequenced completely at Northwoods DNA Inc., Becida, MN. Mpl cDNA were re-cloned from samples that had mutations and sequenced completely. The putative mutations were confirmed by sequencing the corresponding region of the Mpl gene from respective samples. For this, the genomic DNA isolated from PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25636517 the peripheral blood samples were PCR amplified with gene specific primers and the PCR products were directly sequenced after purification using the Wizard PCR Preps DNA Purification System from Promega, Madison, WI.In vitro transcription/translation of Mpl The Mpl cDNA clones in the Blue Script vector were digested with EcoR 1 and Xho 1 and the released Mpl cDNA was cloned into pcDNA 3 vector to obtain cDNA with 3′ translational signals. These constructs were then subjected to in vitro transcription/translation using the TNT Quick Coupled Transcription/Translation Systems from Promega. Madison, WI, in the presence of 35S Methionine from Amersham Biosciences. About 0.5 of the constructs were incubated with the TNT Quick-Coupled Master Mix and 35S Methionine in the absence or presence of 2 of Canine microsomal membranes provided with the TNT Quick Coupled kit, at 30 for 2 hours. The reactions were terminated by adding 100 of 1X SDS loadingMPL FP 5′-AAGTCCTCAGAGAGGACTCCTTTG-3 MPL RP 5′-CAGGCAAGAAGGCTGCAATC-3′ MPL PROBE 5′-FAM CCTCCCAGGCCCAGATGGACTAC3′ BHQ1 The reaction conditions were reverse transcription at 50 for 30′; 40 cycles of amplification with de-naturation at 94 for 15″, annealing and extension at 60 for 10″.Sequence Analysis of Mpl Total RNA extracted from platelets obtained from 15 patients with AMM and 15 normal controls was used to clone the full length Mpl cDNA. First strand cDNA was synthesized using the SuperScript First Strand Synthesis System for RT-PCR from Invitrogen, Carlsbad, CA,Page 8 of(page number not for citation purposes).