2. You do not need to use autoclaved tubes, but tubes exclusively
reserved for RNA purification, and do not manipulate the tubes
without wearing gloves.
3. We recommend using TRIzol™ from Thermo Fisher Scientific
as it has proven to work optimally with this protocol. Another
equivalent commercially available reagent might work with a
comparable efficiency. Alternatively, home-made solutions can
be prepared (see ref. 12, 13, 15), but the process is laborious.
4. Prepare the 75% ethanol solution in a RNase-free conical tube
and discard any leftover.
5. We recommend using the Turbo DNA-free™ turbo kit from
Invitrogen (Thermo Fisher Scientific).
6. If you do not have at your disposal an exclusive designated
laboratory space for RNA extraction, it might be wise to perform RNA purification when there are not too much people in
the laboratory, lowering air perturbation and the risk of dust
movement and contamination. Regardless, the surface and any
equipment used should be cleaned with a surface decontaminant DNA/RNase removing solution. We recommend using
the RNase Away from Merck.
7. If possible, we recommend having a designated pipette set and
gel equipment exclusively used for RNA purification.
8. We cultivate Leptospira in EMJH medium. It is possible to
extract RNA from lower amounts of cells; however, the yield
will be lower, and, in our experience, working with low
amounts of cells leads to an RNA preparation with a lower
integrity. It is not necessary to wash the cells before adding
the guanidinium thiocyanate-acidic phenol solution.
9. The cells should be rapidly processed in the guanidinium
thiocyanate-acidic phenol solution (TRIzol™) after harvesting
them as rapid inactivation of endogenous RNases is essential
for obtaining high-quality RNA.
10. Manipulation of the guanidinium thiocyanate-acidic phenol
solution (TRIzol™) should be done under a fume hood, as
the solution is highly volatile and toxic.
11. Even if RNA extraction is conducted right after cell resuspension in the guanidinium thiocyanate-acidic phenol solution
(TRIzol™), samples should be frozen at À80
C as freezing
promotes cell lysis. Samples can be stored in the guanidinium
thiocyanate-acidic phenol solution (TRIzol™) at À80
C for at
least several weeks. If you plan to analyze RNAs extracted from
different biological samples, it is better to perform the RNA
extraction of all samples at the same time.
12. In order to increase efficiency of cell lysis, up to three cycles of
freezing/thawing can be applied to the samples. However, as
48
Crispin Zavala-Alvarado and Nadia Benaroudj
reserved for RNA purification, and do not manipulate the tubes
without wearing gloves.
3. We recommend using TRIzol™ from Thermo Fisher Scientific
as it has proven to work optimally with this protocol. Another
equivalent commercially available reagent might work with a
comparable efficiency. Alternatively, home-made solutions can
be prepared (see ref. 12, 13, 15), but the process is laborious.
4. Prepare the 75% ethanol solution in a RNase-free conical tube
and discard any leftover.
5. We recommend using the Turbo DNA-free™ turbo kit from
Invitrogen (Thermo Fisher Scientific).
6. If you do not have at your disposal an exclusive designated
laboratory space for RNA extraction, it might be wise to perform RNA purification when there are not too much people in
the laboratory, lowering air perturbation and the risk of dust
movement and contamination. Regardless, the surface and any
equipment used should be cleaned with a surface decontaminant DNA/RNase removing solution. We recommend using
the RNase Away from Merck.
7. If possible, we recommend having a designated pipette set and
gel equipment exclusively used for RNA purification.
8. We cultivate Leptospira in EMJH medium. It is possible to
extract RNA from lower amounts of cells; however, the yield
will be lower, and, in our experience, working with low
amounts of cells leads to an RNA preparation with a lower
integrity. It is not necessary to wash the cells before adding
the guanidinium thiocyanate-acidic phenol solution.
9. The cells should be rapidly processed in the guanidinium
thiocyanate-acidic phenol solution (TRIzol™) after harvesting
them as rapid inactivation of endogenous RNases is essential
for obtaining high-quality RNA.
10. Manipulation of the guanidinium thiocyanate-acidic phenol
solution (TRIzol™) should be done under a fume hood, as
the solution is highly volatile and toxic.
11. Even if RNA extraction is conducted right after cell resuspension in the guanidinium thiocyanate-acidic phenol solution
(TRIzol™), samples should be frozen at À80
C as freezing
promotes cell lysis. Samples can be stored in the guanidinium
thiocyanate-acidic phenol solution (TRIzol™) at À80
C for at
least several weeks. If you plan to analyze RNAs extracted from
different biological samples, it is better to perform the RNA
extraction of all samples at the same time.
12. In order to increase efficiency of cell lysis, up to three cycles of
freezing/thawing can be applied to the samples. However, as
48
Crispin Zavala-Alvarado and Nadia Benaroudj