Reverse transcriptase-polymerase chain reaction (RT-PCR)

First-strand complimentary DNA (cDNA) synthesis was performed using a cDNA synthesis kit (Promega, Madison, WI, USA) according to the manufa­cturer’s protocol. cDNA synthesis was performed by reverse transcription in a total volume of 20 p l reaction mixture containing 1 g RNA, 2 p l of 10xreaction buffer (100 mM Tris-HCl, pH 9.0, 500 mM KCl, 1% Triton X-100), 4 pl of 25 mM MgClz, 2 pl of 10 mM of each deoxynucleotide triphophate (dNTP), 0.5 g random primer, 20 U of RNase inhibitor, and 15 U AMV reverse transcriptase. The mixture was incubated at 25oC for 10 min, 42oC for 1 hr, then heated to 95oC for 5 min and chilled on ice. The PCR reaction was performed in a mixture of 50 p l containing 2 p l cDNA, 5 p l of 10xreaction buffer (100 mM Tris-HCl pH 8.3, 500 mM KCl, 15 mM MgClz), 4 pl of 2.5 mM of each dNTP, 20 pmol of each primer (Table 1), and 2 U Taq DNA polymerase (Bioneer, Korea). All reactions were carried out in a thermal cycler (Perkin Elmer 9600, Norwalk, CT, USA) with an initial dena- turation step of 95oC for 4 min. 30 cycles were then performed of denaturing at 95oC for 1 min, annea­ling at 58oC (for TGF- в 1) and 55oC (for TGF-в 2, TGF^3, and в-actin) for 1 min, and extension at 72oC for 1 min.

Table 1. Oligonucleotide sequences of the primers used for RT-PCR analysis

Gene

Sequences (5′ ^ 3′)

Size of product (bp)

TGF- в 1

Sense

5-GCCCTGGACACCAACTATTGC-3′

333

Antisense

5′-GCACTTGCAGGAGCGCA-3′

TGF-в 2

Sense

5′-AAATGGATACACGAACCCAA-3′

267

Antisense

5′-GCTGCATTTGCAAGACTTTAC-3′

TGF- в 3

Sense

5′-AAGTGGGTCCATGAACCTAA-3′

267

Antisense

5′-GCTACATTTACAAGACTTCAC-3′

в -actin

Sense

5′-TCATGAGGTAGTCAGTCAGG-3′

305

Antisense

5′-CTTCTACAATGAGCTGCGTG-3′

The run was terminated at 72oC for 5 min, and the temperature was then reduced to 4oC for sample storage until further processing. The negative controls—water instead of cDNA and the products of the reverse transcription (RT) reac­tions without reverse transcriptase—were subjected to polymerase chain reaction (PCR) and were confirmed to have no false-positive reactions. 10 p l of PCR products were separated by electrophoresis on a 2% agarose gel containing 0.5 g/ml ethidium bromide and visualized by image analysis (Gel Doc 1,000 gel documentation system, Bio-Rad, Hercules, CA, USA). The size marker was DNA molecular weight marker 100 bp ladder (Takara, Japan), and PCR band intensity was measured by a densitometer (Model GS-700 imaging densitometer, Bio-Rad) and expressed as intensities relative to в-actin. The ex­pression levels of TGF-/s mRNA were measured after 24 hr and 48 hr.
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Statistical analysis

The results were expressed as mean±SD. The statistical significance of differences between the quantity of TGF- в protein and TGF- в mRNA levels were tested using the Student’s t-test, with a p value of less than 0.05 considered significant.