18S/28S RNA in kb
hamel at ccu.umanitoba.ca
Sat Sep 4 10:12:33 EST 1993
In article <CCswyJ.9qJ at ccu.umanitoba.ca> hamel at ccu.umanitoba.ca (Andre Hamel) writes:
>In article <01H2ISR5IVNM001WSM at msvax.mssm.edu> BISHOP at MSVAX.MSSM.EDU ("DAVID F. BISHOP") writes:
>>DA Gray (IN%"gray at labsun1.med.uottawa.ca") writes:
>>> A reviewer has asked for the values we use to estimate
>>> RNA sizes on northerns. There is a folk myth in the
>>> lab that these values are 1.8 kb and 5.0 kb for 18S
>>> and 28S respectively, but like any good folk myth
>>> this one cannot be documented. Does anyone have a
>>> reference for the actual sizes of eukaryotic rRNAs?
>>> I would be grateful for the help.
>Ribosomal RNA bands vary in size depending upon species (genus more
>likely?) of organism ...
>Take a look in Winter 1993 (Vol. 15, No. 1) issue of BRL Focus, "Rapid
>Micro- Scale Isolation of RNA for PCR or Translation", D.Simms, pgs 6- 11.
>On page 8 and 9 there's 2 nice photos of RNA gels (1.5% agarose/1.1%
>formaldehyde/MOPS) with RNA size standards ... clearly showing 18S and 28S
>rRNAs as about 2.2 and 4.0 kb respectively.
Also, APPARENT size of RNA can be dependent upon electrophoresis
conditions ... hence differences can be seen if separating in different
buffers, with/without "denaturants" such as formaldehyde, methylmercury,
etc. (I often just run my RNA in 1.5% agarose in 1x TBE, pH 8.3 (rRNAs can
run more slowly in denaturing conditions, hence appear as 2.2 and 4.0 kb
for 18S & 28S ... whereas can appear as smaller sizes in non-denaturing
conditions) ... denaturing gel I use is horizontal (vertical gives
sharper bands though) 1.5% agarose in 1x TAE, pH 7.0/1-2% formaldehyde in
gel AND in tank buffer ... RNA preheated 65C/5 min in at least 50%
formamide with about 100 ng EtBr, tracking dye (BPB, NO xylene cyanole,
elsewise RNA can "die"), then sit on ice or load immediately ... another
nice "sharp resolution" type gel consists of "regular" 7% acrylamide/0.3%
bis/7 M Urea/1x TBE, pH 8.3 type sequencing gel mix (fresh made ... DON'T
store acryl/bis/Urea in TBE for longer than few days), but one doesn't need
50 cm gels for northerns ... 0.7 mm spacers in 15 x 15 cm plates is enough
size for good "spread" between 500 nt to about 10 kb (also, if RNA size
standards not available, RE. digested DNA stds llikewise heat treated in
formamide/EtBr work nice .... a caveat is that ssDNA migrates slightly
different than ssRNA ("a tad" more slowly).
Andre Hamel email: hamel at ccu.umanitoba.ca
Manitoba Veterinary Services lab tel.: (204) 945-7630
Infectious & Genetic Disease FAX: (204) 945-8062
Mol.Biol.Lab., Winnipeg, Manitoba, CANADA
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