3.3.2 Quality Control
for Cell Sorting by
DNA-PCR
As mentioned above, total RNA extracted from the “mix” and “coculture” samples contains genomic DNA contaminants, as they
have not been treated with DNase I. Therefore, DNA amplification
by PCR can be readily used to check the efficiency of sorting, in
addition to flow cytometry method described above.
1. Choose test amplicons for genes specific to the different cell
types. For example, in the co-culture system of MBS-MEF and
MCF7 cells, we use forward and reverse primers that can
specifically detect either the MBS-3
0 UTR junction sequence
of the β-actin-MBS gene (present only in MBS-MEF cells) or
the human MALAT1 (present only in MCF7 cells). Thus, we
expect that the purified mouse cell fractions should contain
only the β-actin-MBS band (and not MALAT1), while purified
human cell fractions should have only the MALAT1 band (and
not β-actin-MBS). Presence of MALAT1 in the mouse cell
fraction and β-actin-MBS in the human cell fraction indicates
that the samples are not pure. This may result in a high occurrence of nonspecific reads during deep sequencing (see
Note 10).
2. Set up the PCR reaction in a 20 μL reaction volume using
gene-specific primer pairs described above (i.e., ~20 bp each,
50–60% GC content and melting temperature 55–60
C), as
recommended by the manufacturer of PCR reaction mix
employed. The following calculation for reaction mix can be
used as a reference: premade PCR mix—at a concentration
recommended by the manufacturer; forward and reverse primers—final concentration of 0.8 μM; and genomic DNA template—1 μL; fill the rest of the reaction volume with nucleasefree water to 20 μL.
3. Perform the reaction according to the following conditions:
melting—95
C for 30 s; annealing—55–60
C for 1 min;
elongation—72
C for 1 min; and number of recommended
cycles—less than 30. We recommend 28 cycles to reduce the
nonspecific amplification.
4. Check the amplification results by running the reaction in a 1%
(w/v) agarose gel for 20 min at 200 V prior to staining with
ethidium bromide. See Fig. 3b for representative results.
ä
Fig. 3 (continued) gene is used to test for MBS-MEFs (Fig. 1) and a human specific long noncoding RNA gene,
MALAT1, for MCF7 cells). 18S rRNA was amplified as an internal control. As expected, the β-actin-MBS band
was detected only in mouse cells, while MALAT1 was detected only in human cells, indicating a high degree of
purity of magnetic sorting. Blue boxes—MBS-MEF mouse cells, red boxes—MCF7 human cells
Use of RNA Tagging as a Control for RNA Transferome Analysis
205
for Cell Sorting by
DNA-PCR
As mentioned above, total RNA extracted from the “mix” and “coculture” samples contains genomic DNA contaminants, as they
have not been treated with DNase I. Therefore, DNA amplification
by PCR can be readily used to check the efficiency of sorting, in
addition to flow cytometry method described above.
1. Choose test amplicons for genes specific to the different cell
types. For example, in the co-culture system of MBS-MEF and
MCF7 cells, we use forward and reverse primers that can
specifically detect either the MBS-3
0 UTR junction sequence
of the β-actin-MBS gene (present only in MBS-MEF cells) or
the human MALAT1 (present only in MCF7 cells). Thus, we
expect that the purified mouse cell fractions should contain
only the β-actin-MBS band (and not MALAT1), while purified
human cell fractions should have only the MALAT1 band (and
not β-actin-MBS). Presence of MALAT1 in the mouse cell
fraction and β-actin-MBS in the human cell fraction indicates
that the samples are not pure. This may result in a high occurrence of nonspecific reads during deep sequencing (see
Note 10).
2. Set up the PCR reaction in a 20 μL reaction volume using
gene-specific primer pairs described above (i.e., ~20 bp each,
50–60% GC content and melting temperature 55–60
C), as
recommended by the manufacturer of PCR reaction mix
employed. The following calculation for reaction mix can be
used as a reference: premade PCR mix—at a concentration
recommended by the manufacturer; forward and reverse primers—final concentration of 0.8 μM; and genomic DNA template—1 μL; fill the rest of the reaction volume with nucleasefree water to 20 μL.
3. Perform the reaction according to the following conditions:
melting—95
C for 30 s; annealing—55–60
C for 1 min;
elongation—72
C for 1 min; and number of recommended
cycles—less than 30. We recommend 28 cycles to reduce the
nonspecific amplification.
4. Check the amplification results by running the reaction in a 1%
(w/v) agarose gel for 20 min at 200 V prior to staining with
ethidium bromide. See Fig. 3b for representative results.
ä
Fig. 3 (continued) gene is used to test for MBS-MEFs (Fig. 1) and a human specific long noncoding RNA gene,
MALAT1, for MCF7 cells). 18S rRNA was amplified as an internal control. As expected, the β-actin-MBS band
was detected only in mouse cells, while MALAT1 was detected only in human cells, indicating a high degree of
purity of magnetic sorting. Blue boxes—MBS-MEF mouse cells, red boxes—MCF7 human cells
Use of RNA Tagging as a Control for RNA Transferome Analysis
205