MACHEREY-NAGEL – 03/2021, Rev. 05
6
RNA clean up XS
2
Product description
2.1 The basic principle
A major aspect of RNA clean up is preventing degradation of the RNA during the clean
up procedure. The
NucleoSpin
®
RNA Clean-up XS
method achieves this by mixing the
crude RNA extract with a binding buffer, containing chaotropic ions, and ethanol. This buffer
immediately inactivates RNases (which are present in virtually all biological materials) and
creates appropriate binding conditions to allow adsorption of RNA to the silica membrane.
Two washing steps with a single buffer remove any impurities. Pure RNA is finally eluted at
low ionic strength conditions with RNase-free water (supplied) in a volume as small as 5 µL.
The RNA clean up procedure using
NucleoSpin
®
RNA Clean-up XS
kit can be performed
at room temperature. The eluate should be treated with care because RNA is very sensitive
to trace contaminations of RNases, often present on general lab ware, fingerprints, and dust.
To ensure RNA stability, we recommend keeping the RNA solution frozen at -20 °C for short-
term or -70 °C for long-term storage.
2.2 Kit specifications
• The
NucleoSpin
®
RNA Clean-up XS
kit is recommended for the clean up and
concentration of prepurified RNA samples. Typical sample material covers nanogramm
to microgramm amounts of prepurified RNA (e.g., phenol-purified RNA) and RNA from
reaction mixtures (e.g., DNase treated samples).
• The innovative column design with a funnel shaped thrust ring and a small silica
membrane area allows sample volumes of up to 300 µL and elution of RNA in as
little as 5–30 µL. Thus,
highly concentrated RNA
is eluted and is ready for common
downstream applications (e.g., RT-PCR). RNA enrichment of 20 x up to 50 x can be
achieved (e.g., input: 300 µL sample containing crude RNA (10 ng/µL); output: 5 µL
eluate containing pure RNA (510 ng/µL); enrichment of factor 51 (MACHEREY-NAGEL
in-house data)).
• The
RNA recovery rate
is typically 85–95 %.
• High quality RNA (RNA Integrity Number (RIN) > 9 according to Agilent 2100
Bioanylzer assays) can be obtained from high quality RNA samples. The RIN of the
processed sample is typically equal (±0.3) to the RIN of the input sample. RNA quality
always depends on the sample quality, see section 6.3 for further aspects.
• The
NucleoSpin
®
RNA Clean-up XS
kit allows clean up and concentration of RNA
with an A
260
/A
280
ratio generally exceeding 1.9 (measured in TE buffer pH 7.5). Due
to the high RNA purity, large amounts of eluates can be used as template in RT-PCR
without inhibition (e. g., 8 µL of 10 µL eluates as template in a 20 µL qRT-PCR setup
generating stronger signal compared to reactions with less template in a LightCycler™
PCR with the Sigma SYBR
®
Green Quantitative RT-PCR Kit).
