Analysis of BMP-6 messenger RNA (mRNA) expression
Endogenous expression of the BMP-6 gene was examined by reverse transcription-polymerase chain reaction. Total RNA was isolated using Absolutely RNAtrade RT-PCR Miniprep Kit (Stratagene Europe, Amsterdam, Netherland) according to the manufacturers instructions. Quantification of the isolated total RNA was achieved by using spectrophotometric OD260 measurements. Equal amounts of RNA were then reverse transcribed to cDNA with TaqMan(R) Reverse Transcription Reagents (Applied Biosystems, Foster City, CA, U.S.A). To measure mRNA expression of BMP6, Id1-Id4 and PGK1 PCR were carried out with TaqMan(R) universal master mix. Primers and probes were provided by Assay-on-Demand (Applied Biosystems). PCR reactions were carried out in a final volume of 25 mul (BMP-6) or 20 mul (ID1). The cDNA added to each reaction was equivalent to the input of 20 ng of total RNA. The gene expression was quantified using the standard curve method (BMP6), or the comparative CT method (Id1) as described in ABI7700 User Bulletin 2 (Applied Biosystems). The expression was then normalized to the expression level of PGK1. PGK1 was chosen, because it has been shown to have low expression variability among lymphocyte specimens [56]. Expression levels in B cells were then related to the expression levels in Ramos cells. 
