15
MACHEREY-NAGEL – 03/2021, Rev. 05
RNA clean up XS
Problem
Possible cause and suggestions
Suboptimal
performance
of RNA in
downstream
experiments
Carry-over of ethanol or salt
• Do not let the flowthrough touch the column outlet after the
second wash using Wash Buffer RA3. Be sure to centrifuge at the
corresponding speed for the respective time in order to remove
ethanolic Wash Buffer RA3 completely.
• Check if Wash Buffer RA3 has been equilibrated to room
temperature before use. Washing at lower temperatures lowers
efficiency of salt removal by Wash Buffer RA3.
• Depending on the robustness of the used RT-PCR system, RT-
PCR might be inhibited if complete eluates are used as template
for RT-PCR. Use less eluate as template.
Store isolated RNA properly
• Eluted RNA should always be kept on ice for optimal stability since
trace contaminations of omnipresent RNases (general lab ware,
fingerprints, dust) will degrade the isolated RNA. For short term
storage freeze at -20 °C, for long term storage freeze at -70 °C.
Higher RNA
yield than
theoretically
possible
• If performing clean up of samples containing less than
approximately 300 ng RNA, subsequent quantification by A
260
measurement may simulate yields larger than the RNA input. This
may be due to absorbance of silica abrasion. In order to prevent
incorrect A
260
quantification of small RNA amounts, centrifuge the
elution tube for 30 s at 8.000–11.000 x
g
and withdraw an aliquot
for measurement without disturbing any sediment or use a silica
abrasion insensitive RNA quantification method (e.g., RiboGreen
®
fluorescent dye).
Unexpected
A
260
/A
280
ratio
Measurement not in the range of photometer detection limit
• In order to obtain a significant A
260
/A
280
ratio it is necessary that
the initially measured A
260
and A
280
values are significantly above
the detection limit of the photometer used. An A
280
value close to
the background noise of the photometer will cause unexpected
A
260
/A
280
ratios.
