2. Add 1 μl of Turbo DNA-free™ DNase (at 2 U/μl). Mix by
pipetting and incubate 30 min in a water bath at 37
C (see
Notes 20 and 21).
3. Add 5 μl of DNase inactivation reagent (provided in the kit),
mix well by flicking the tube to disperse the inactivating
reagent, and incubate for 5 min at room temperature. During
this incubation, flick the tube to disperse the inactivating
reagent each minute in order to increase the binding of the
DNase to the reagent (see Note 22).
4. Centrifuge for 2 min at 10,000 Â g and transfer the supernatant to a new clean polypropylene tube (see Note 23).
3.4 Assessing
Quantity and Quality
of RNA
The absorbance measurement at 260 nm allows the calculation of
RNA concentration. An absorbance value of 1 corresponds to
40 μg/ml of RNA (for a spectrophotometer with 1 cm light path).
For RT-PCR, the quality of the RNA preparation can be
assessed by electrophoresis on an agarose gel. When 0.5–1 μg of
RNA are loaded on a 1% agarose gel, three main bands can be
observed, the 23S, the 16S, and the 5S ribosomal RNAs (Fig. 1)
as the total RNA preparation contains mainly ribosomal RNAs.
Messenger RNAs can be sometimes visible as faint smear.
If the RNA is to be used in RNA-Seq, the integrity of RNA
should be assessed by capillary electrophoresis (see Note 24). A
typical electrophoresis pattern of high-quality RNA is shown in
Fig. 1 Analysis of RNA preparation on agarose gel. 0.5 μg of total RNAs were
loaded on a 1% agarose gel in 1ÂTAE. Nucleic acid was stained with ethidium
bromide. The bands corresponding to 23S, 16S, and 5S ribosomal RNA are
indicated
46
Crispin Zavala-Alvarado and Nadia Benaroudj
pipetting and incubate 30 min in a water bath at 37
C (see
Notes 20 and 21).
3. Add 5 μl of DNase inactivation reagent (provided in the kit),
mix well by flicking the tube to disperse the inactivating
reagent, and incubate for 5 min at room temperature. During
this incubation, flick the tube to disperse the inactivating
reagent each minute in order to increase the binding of the
DNase to the reagent (see Note 22).
4. Centrifuge for 2 min at 10,000 Â g and transfer the supernatant to a new clean polypropylene tube (see Note 23).
3.4 Assessing
Quantity and Quality
of RNA
The absorbance measurement at 260 nm allows the calculation of
RNA concentration. An absorbance value of 1 corresponds to
40 μg/ml of RNA (for a spectrophotometer with 1 cm light path).
For RT-PCR, the quality of the RNA preparation can be
assessed by electrophoresis on an agarose gel. When 0.5–1 μg of
RNA are loaded on a 1% agarose gel, three main bands can be
observed, the 23S, the 16S, and the 5S ribosomal RNAs (Fig. 1)
as the total RNA preparation contains mainly ribosomal RNAs.
Messenger RNAs can be sometimes visible as faint smear.
If the RNA is to be used in RNA-Seq, the integrity of RNA
should be assessed by capillary electrophoresis (see Note 24). A
typical electrophoresis pattern of high-quality RNA is shown in
Fig. 1 Analysis of RNA preparation on agarose gel. 0.5 μg of total RNAs were
loaded on a 1% agarose gel in 1ÂTAE. Nucleic acid was stained with ethidium
bromide. The bands corresponding to 23S, 16S, and 5S ribosomal RNA are
indicated
46
Crispin Zavala-Alvarado and Nadia Benaroudj