Total cellular RNA was extracted from rabbit lacrimal gland tissue
using TRIzol (Gibco BRL, Rockville, MD) according to the
manufacturer’s instructions. RNA was dissolved in diethyl procarbonate
water, and the concentrations were determined using a
spectrophotometer. A cDNA probe for rabbit HGF, KGF, EGF, or β-actin
was amplified using polymerase chain reaction (PCR) with the primers
listed in
Table 1 . A melting temperature of 98°C, an annealing temperature of 55°C,
and an extension temperature of 72°C were used in PCR amplifications,
which were continued for 35 cycles. The amplification products were
cloned into the pCR2.1 TA clone vector (Invitrogen, San Diego, CA) and
sequenced using standard methods to confirm that the sequence was the
corresponding rabbit sequence for each cytokine or β-actin. In some
cases, sequences were subcloned into the Bluescript SK+ vector
(Stratagene, San Diego, CA) so that the antisense RNA probe used in the
RNase protection assay would be synthesized from the T7 promoter,
whether the sequence was in the pCR2.1 TA or Bluescript vector. A
32P-labeled RNA probe for each cytokine and β-actin was
prepared with an RNA transcription kit (Stratagene).
The RNase protection assay was performed with a kit (Boehringer
Mannheim, Indianapolis, IN), according to the manufacturer’s protocol
with the following modifications: Two microliters of RNA probe (1 × 106 cpm/μl) and 50 μg of rabbit lacrimal gland RNA
(100 μg RNA was used for the KGF experiment) were coprecipitated in
the presence of 0.3 M sodium acetate with ice-cold ethanol. β-Actin
RNA probe (1 × 108 cpm/μl) was included with each
growth factor RNA as a control for quantitation. RNA was recovered by
centrifugation at 15,000 rpm for 15 minutes at 4°C in a
microcentrifuge and dissolved in 30 μl hybridization buffer. After
denaturation for 5 minutes at 90°C, the samples were incubated
overnight at 42°C in an oven. Each hybridization mixture was digested
with 40 units of RNase T1 and 10 units of RNase T2 for 50 minutes at
30°C, then digested with 3 μl proteinase K (20 μg/μl) in the
presence of 0.5% sodium dodecyl sulfate (SDS) for 20 minutes at
37°C. The protected RNA fragments were precipitated by adding 5 μg
yeast tRNA and 1 ml ethanol and then extracted with 400 μl
phenol-chloroform (1:1). The RNA pellet was resuspended in 7 μl
loading buffer, heated for 5 minutes at 90°C, and subjected to
electrophoresis in a 4% polyacrylamide–7 M urea gel at 300 V in 1×
TBE (89 mM Tris-HCl, 89 mM sodium borate, 2 mM EDTA) buffer. The gel
was fixed with 10% acetic acid and 10% methanol and dried with a
vacuum gel dryer (Bio-Rad, Richmond, CA). Dried gels were exposed
overnight to film (i; Eastman Kodak, Rochester, NY). Unless otherwise
specified, all reagents were obtained from Sigma (St. Louis, MO).
The actual sizes of the protected RNA fragments were confirmed using 32P-labeled RNA ladder that was included on each gel. This
ladder was prepared by using the full-length transcripts from the
cloned cDNA templates for each of the growth factors and β-actin. The
full-length RNA probe sizes (not protected sequence size) were 320
(HGF), 460 (β-actin), 480 (KGF), and 550 (EGF) nucleotides. These
markers were revealed with a brief exposure, and then the marker lane
was cut from the dried gel to prevent overexposure of adjacent
RNase-protected lanes.
Quantitation of bands was performed by scanning the gels (Photoshop
4.0; Adobe, Seattle, WA) and using NIH Image 1.57 software to determine
the density of both the growth factor and β-actin band in each lane.
All lanes were run on gels simultaneously for a particular
RNase-protection experiment. A uniform box size that enclosed the
largest band in a series for a particular growth factor or β-actin
was used for determining all the growth factor or β-actin band
densities in a particular experiment. The relative intensity of each
band was calculated as density units = (density of growth factor
band/density of β-actin band) × 100. There were four different
animals at each time point after wounding and in the control.
Statistical analyses were performed with analysis of variance using the
Bonferroni–Dunn adjustment. P < 0.05 was
considered statistically significant.