【请教】引物的TM值太高影响RNA探针的原位杂交吗？

刚开始作RT-PCR,让同学帮我设计了三个基因的上下游引物，如下： Primer A (bp233C): 5'-GAATTCACCACACGGCCATTGAAGCA-3'
Primer B (bp25): 5'-AAGCTTCACATGGACGAGGAGGAGAC-3'

Primer Balance: -A- -B- Comment
Length 26 26
% GC 50 53 3 % difference
Tm(癈) 78 71 7 C?difference
Tm (2nd round) 84 80 4 C?difference

Primer Cautions:
Stability (kcals) 3.5 3.1
Runs of bases 2 2
Repeats (dinuc) 2 2
Hairpins none none

Primer Dimers: A:A B:B A:B
3' end matches 2 1 2
Any adj homol bases 6 6 6

Primer A (bp275C): 5'-GAATTCGACGACACAAGCCAGGAGAC-3'
Primer B (bp31): 5'-AAGCTTGTAGGCGAGACGAAGGTGAT-3'

Primer Balance: -A- -B- Comment
Length 26 26
% GC 53 50 3 % difference
Tm(癈) 72 70 2 C?difference
Tm (2nd round) 81 79 2 C?difference

Primer Cautions:
Stability (kcals) 3.0 4.2
Runs of bases 2 2
Repeats (dinuc) 2 2
Hairpins none none

Primer Dimers: A:A B:B A:B
3' end matches 1 2 2
Any adj homol bases 6 6 6

Primer A (bp340C): 5'-GAATTCGAACTACGGCCAGATAGTGC-3'
Primer B (bp98): 5'-AAGCTTAGCTGCTCATCCTGCTGTTG-3'

Primer Balance: -A- -B- Comment
Length 26 26
% GC 50 50
Tm(癈) 68 72 4 C?difference
Tm (2nd round) 78 79 1 C?difference

Primer Cautions:
Stability (kcals) 3.9 2.1
Runs of bases 2 2
Repeats (dinuc) 1 1
Hairpins none none

Primer Dimers: A:A B:B A:B
3' end matches 2 2 2
Any adj homol bases 6 6 3
发现这三对引物的TM值都挺高，尤其第一对引物之间的TM值相差挺大。这会影响RT-PCR结果吗？我后面还要用此PCR扩增参物作RNA探针用于原位杂交，这么高的TM值有影响马？恳切等候各位高手的答案。先谢了。。。