Figure 1 A shows the 228-bp and
Figure 1B the 373-bpβ
B
2-crystallin products generated with primer
pairs 4 and 3
(Table 1) , respectively. Two and 10 μg total RNA (1×
and 5×) were used in the RT-PCR procedure. With primer set 4
(Fig. 1A) , βB
2-crystallin mRNA was detected in brain
(lanes 1 and 2), testis (lanes 3 and 4), and as control lens (lane 5).
With primer set 3
(Fig. 1B) , very low amounts ofβ
B
2-crystallin mRNA were detected in brain
(lanes 6 and 7), whereas high levels ofβ
B
2-crystallin mRNA were detected in testis
(lanes 8 and 9) and lens (lane 10). To further confirmβ
B
2-crystallin expression in these tissues,
fresh total RNA samples were prepared from 60- to 90-day-old rat lens,
brain, lung, heart, testis, ovary, spleen, thymus, kidney, and liver
and analyzed by RT-PCR with additional primer sets (
Table 1 , oligo sets
1 and 2). Significant amounts of βB
2-crystallin
were detected in brain, lens, and testis total RNA (
Fig. 1C , lanes 1
and 2;
Fig. 1D , lane 1). The lens band appeared weaker than that found
for brain. This is likely the result of minor degradation of this
sample as evidenced by the presence of a lower band in the lens lane
(
Fig. 1C , lane 1) and reduced levels of the corresponding β-actin
band (
Fig. 1C , lane 2). All products were reverse
transcriptase-specific, and representative control products from brain
and lens were sequenced and confirmed to be authenticβ
B
2-crystallin (data not shown). β-Actin
transcript was detected at high levels in corresponding reactions (
Fig. 1C , lanes 3 and 4;
Fig. 1D , lane 2).β
B
2-crystallin mRNA was not detected by RT-PCR
in 1.0 μg (
Table 1 , oligo set 1) or 1.4 μg (
Table 1 , oligo set 2)
total RNA from lung, heart, ovary, spleen, thymus, kidney, or liver
(data not shown).