Total RNA was isolated from the keratinized conjunctivas by the
use of reagent (Trizol; Gibco) in accordance with the manufacturer’s
protocol. To investigate relative levels of
TGase 1 mRNA
expression in diseased conjunctiva, semiquantitative reverse
transcription–polymerase chain reaction (RT-PCR) was
performed.
35 The human
G3PDH gene was used as
the internal control. Primer sequences used were ACCACAGTCCATGCCATCAC
(sense) and TCCACCACCCTGTTGCTGTA (antisense). cDNA was generated by
mixing the extracted RNA after ethanol precipitation (1 μg/μl per
sample) with a random hexamer primer (Takara Biomedicals, Tokyo, Japan)
and incubating at 65°C for 5 minutes, while chilling the samples on
ice. The mixture was then subjected to reverse-transcription in 25 mM
MgCl
2, 100 mM Tris-HCl (pH 8.3), 500mM KCl, 40
U/μl RNase inhibitor (Takara Biomedicals), 10 mM dNTP mixture, and 5
U/μl reverse transcriptase (AMV-XL, final volume, 20 μl; Takara
Biomedicals). The mixture was incubated at 30°C for 10 minutes and at
42°C for 30 minutes, heated to 99°C for 5 minutes, and then stored
at −20°C until use. A 10-μl aliquot (half of the total volume) of
the same RT product (per sample) was used for PCR amplification.
Oligonucleotide primers to the nontandem repeat regions of
TGase1 were designed from published
36 or GenBank sequences CCTTCTGGGCTCGCTGCTGTGG (sense) and CCACGAGAGCCGCCAAGACCAG (antisense). PCR amplifications were performed
as previously described,
37 with conditions optimized for
the
TGase1 gene using the RT product from total conjunctival
RNA. The linear range of the amplification reaction for
TGase
1 and
G3PDH was determined by checking amplification
after each cycle from cycles 23 to 30 for
TGase 1 and from 21 to 31 for
G3PDH. This showed that the 27th cycle
was in the midlinear phase for
TGase 1 and
G3PDH.
All PCR amplifications started with denaturation at 95°C for 3
minutes and ended with a final elongation at 72°C for 10 minutes. The
parameters for PCR amplification were as follows: 27 cycles of
denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute,
and extension at 72°C for 1 minute. A 5-μl aliquot of the reaction
mixture was then electrophoresed on a 2% agarose gel (Seakem; FMC,
Rockland, ME) containing ethidium bromide to evaluate amplification and
fragment size. The amount of amplified product was quantified for each
sample using a computing densitometer (420OE scanner; PDI, NY)
and software (Quantity One; PDI, NY). The final amount of PCR product
was expressed as the ratio of the
TGase1 gene amplified to
that of the
G3PDH gene, to account for any differences in
beginning amounts of RNA.