RNA extraction from liver tissue and analysis of transcript expression of TGF-, TNF-
 by quantitative RT-PCR
Total RNA was extracted from frozen liver tissue using a commercially available reagent
(Qiagen Rneasy kit (Qiagen GmbH, Hilden, Germany). For quantitative competitive
reverse transcription polymerase chain reaction (RT-PCR) an internal standard (pHCQ1:
840 nt) constructed to be recognized by and to compete for oligonucleotide cytokine
primers was used. Sense and antisense PCR primer sequences were the following:
Forawrd: 5´-ACCGCAACAACGGCCATCTAT-3´
and Reverse: 5´-GTAACGCCAGGAATTGTTGC-3´ for TGF-B1;
Forward: 5'- CAA CTG GAG CAT TTA CTG GA-3' and
Reverse: 5'-TCA GTT CTG TGG CTT CTC TTG -3' for TNF-
Forward: 5´-TGGAATCCTGTGGCATCCATGAAAC-3´ and
Reverse: 5´-TAAAACGCAGCCTCAGTAACAGTCCG-3´ for b-actin (GibcoBRL, Life
Technologies AG, Basel, Switzerland).
Co-amplification of target complementary DNA (cDNA) corresponding to 50 ng of
cellular RNA and internal standard was performed for 34 cycles for TGF-, 35 cycles
TNF- and 26 cycles for b-Actin with an initial denaturation step for 20 seconds at 94°C;
annealing for 20 seconds at 53.5°C (TGF-) and 72 °C (TNF-) and extension for 30
seconds at 72°C. PCR products were electrophoresed on an ethidium bromide gel and
visualized using ultraviolet light. The amplification products for different cytokines were
for TGF-: standard 246 and target 161; TNF-: standard 432 and target 355, -actin
standard 384 and target 501. Titers were expressed as the last dilution giving a visible
band of the appropriate size on a 2% agarose gel stained by ethidium bromide.
Intrahepatic TGF- RNA titers were normalized to an arbitrary -actin mRNA titer of
1,024, as measured on the same specimen. The intrahepatic -actin titer was expressed as
the last fourfold dilution giving a visible band by ethidium bromide staining of RT-PCR
products obtained from 100 ng of total liver RNA and run on a 2% agarose gel.
Measurement of mRNA gene expression with TaqMan (TM) quantitative real-time RTPCR.
Total RNA was reverse transcribed into cDNA using random hexamer and
oligo(dT) primers and Applied Biosystems RT-PCR reagents. Quantitative real-time
fluorescent PCR of the resultant cDNA was performed in an Applied Biosystem 5700
Sequence Detection System using gene specific primers designed using the Primer
Express Software (TM) (Applied Biosystems/Perkin Elmer) and screened for specific
product annealing by BLAST analysis against the GenBank.. Forward and Reverse
primers were synthesized by SIGMA/GENOSYS. YKL40 probe was synthesized by
Biosearch Technologies incorporating 5' 6-FAM fluorophore and 3' Black Hole
Quencher. Actin TaqMan probe and primer pre-developed assay reagents were obtained
from Applied Biosciences and gene specific FRET quenched oligonucleotide TaqMan
probe. The fluorescence of the PCR reaction increases as the specific amplified product
accumulates. The relative amounts of the starting gene template is estimated by the PCR
cycle number at which the fluorescence exceeds the threshold value (Ct). Changes in
gene expression level relative to untreated control sample can be estimated by
normalization to an internal standard housekeeping gene (- actin). Absolute copy
numbers of mRNA are quantitated by comparison to a standard curve of relating the
threshold Ct value to the absolute starting copy number of the gene of interest.