Afterwards, a probe mix was prepared, containing 1 μM of every oligonucleotide of each probe pair.
Briefly, RNA samples (200 ng total RNA) were first reverse-transcribed using a gene-specific probe mix.
The expression of surfactant protein (SP -A, SP -A SP-B, and SP-C; receptor for andanced glycation end-products (RAGE); keratinocyte growth factor (KGF); and beta-actin was analyzed by receptore PCR using a LC480 Probe Master mix (Roche Diagnostics Japan).
PCR was performed in 8 well strips and specific amplification was detected using a TaqMan probe master mix according to the manufacturer's standard protocol.
RNA was isolated using cells-to-ct kit (Applied Biosystems) and the expression of various genes were measured using TaqMan primer probe mix (Applied Biosystems) on an ABI 7900 realtime PCR machine.
DNA was extracted from leaf tissue using a QIAGEN Plant Mini Kit QIAGENN, Valencia, California, USA) and enriched for microsatellites using three probe mixes [Mix 2 = (AG 12, (TG 12, (AAC 6, (AAG 8, (AAT 12, (ACT 12, (ATC 8; Mix 3 = (AAAC 6, (AAAG 6, (AATC 6, (AATG 6, (ACAG 6, (ACCT 6, (ACTC 6, (ACTG 6; Mix 4 = (AAAT 8, (AACT 8, (AAGT 8, (ACAT 8, (AGAT 8] as in Glenn and Schable (2005).
This was either performed through a clinical BRCA test by the local centre, or was undertaken by ourselves by sequencing genomic DNA through the 24 coding exons and intron exon boundaries of BRCA1 and undertaking MLPA using probe mix P002.
Mulitplex ligation-dependent probe amplification reactions using probe mix SALSA P188 MLPA KIT 22q13 Lot 0407, 0906 were performed on 100 ng genomic DNA from 94 IMGSAC multiplex affected individuals, 76 Italian singleton affected individuals and one positive control individual harbouring a known SHANK3 deletion of 100 kb (DNA kindly provided by Dr. Maria Clara Bonaglia).
Briefly, galactose is oxidized using a galactose probe, an enzyme mix, and HRP, generating a colored product analyzed at the optic density of 570 nm.
GFP siRNA were probed using a mix of 10 different DNA oligonucleotides specific to either the sense or antisense GFP sequence.
