3.4 RT-PCR
Verification of RNA
Transfer (Optional)
If a validated RNA which is known to undergo transfer is available,
it can be used as a positive control for the experiment. For example,
in the co-culture system of MBS-MEF cells and MCF7 cells, we
know that β-actin-MBS mRNA transfers from mouse to human
cells. Using sm-FISH, we detected about 30 copies of β-actin-MBS
mRNA (~2% of donor mRNAs) that are transferred from
MBS-MEF to MCF7 by 12 h of co-culture (Fig. 4). Thus, the
transferred β-actin-MBS mRNA is expected to be present in the
human cell fraction after sorting post-co-culture. The transfer can
be verified using reverse transcription and polymerase chain reaction (RT-PCR) with specific primers to detect the presence of
β-actin-MBS mRNA.
1. Treat 1–5 μg of total RNA with about 2 Units DNase I in an
appropriate buffer for 30 min at 37
C (or as per the manufacturer’s instructions).
2. Perform reverse transcription to produce the cDNA by using
1–5 μg of DNase-treated total RNA and random hexamers as
provided in the cDNA preparation kit, as per the manufacturer’s protocol.
3. Set up the PCR reaction in a 20 μL reaction volume with
primers specific for the gene whose mRNA is known to be
transferred (i.e., ~20 bp each, 50–60% GC content and melting
temperature 55–60
C) as recommended by the manufacturer
of the PCR mix. Forward and reverse primers can be used at a
final concentration of 0.8 μM per 2 μL from the reversetranscribed RNA used as template.
4. 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—23–28. We recommend performing the PCR reaction
in the semiquantitative range to reduce the amplification of
nonspecific bands (see Note 11).
5. 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. 5 for representative results.
3.5 Bioanalyzer
Analysis and Deep
Sequencing
Prior to sending RNA samples for deep sequencing, it is highly
recommended to check the quality and integrity of the RNA samples using a bioanalyzer. We use and recommend the Agilent
TapeStation 2200 system, as per the manufacturer’s recommended
protocol. The output of the analysis is in the form of the RNA
Integrity Number (RIN). Typically, RIN scores of >8 are good for
sequencing experiments.
Finally, for the sequencing of the RNA samples, it is recommended to choose a library preparation kit, sequencing kit, and
platform based on the overall objective of the experiment. In
206
Sandipan Dasgupta and Jeffrey E. Gerst
Verification of RNA
Transfer (Optional)
If a validated RNA which is known to undergo transfer is available,
it can be used as a positive control for the experiment. For example,
in the co-culture system of MBS-MEF cells and MCF7 cells, we
know that β-actin-MBS mRNA transfers from mouse to human
cells. Using sm-FISH, we detected about 30 copies of β-actin-MBS
mRNA (~2% of donor mRNAs) that are transferred from
MBS-MEF to MCF7 by 12 h of co-culture (Fig. 4). Thus, the
transferred β-actin-MBS mRNA is expected to be present in the
human cell fraction after sorting post-co-culture. The transfer can
be verified using reverse transcription and polymerase chain reaction (RT-PCR) with specific primers to detect the presence of
β-actin-MBS mRNA.
1. Treat 1–5 μg of total RNA with about 2 Units DNase I in an
appropriate buffer for 30 min at 37
C (or as per the manufacturer’s instructions).
2. Perform reverse transcription to produce the cDNA by using
1–5 μg of DNase-treated total RNA and random hexamers as
provided in the cDNA preparation kit, as per the manufacturer’s protocol.
3. Set up the PCR reaction in a 20 μL reaction volume with
primers specific for the gene whose mRNA is known to be
transferred (i.e., ~20 bp each, 50–60% GC content and melting
temperature 55–60
C) as recommended by the manufacturer
of the PCR mix. Forward and reverse primers can be used at a
final concentration of 0.8 μM per 2 μL from the reversetranscribed RNA used as template.
4. 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—23–28. We recommend performing the PCR reaction
in the semiquantitative range to reduce the amplification of
nonspecific bands (see Note 11).
5. 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. 5 for representative results.
3.5 Bioanalyzer
Analysis and Deep
Sequencing
Prior to sending RNA samples for deep sequencing, it is highly
recommended to check the quality and integrity of the RNA samples using a bioanalyzer. We use and recommend the Agilent
TapeStation 2200 system, as per the manufacturer’s recommended
protocol. The output of the analysis is in the form of the RNA
Integrity Number (RIN). Typically, RIN scores of >8 are good for
sequencing experiments.
Finally, for the sequencing of the RNA samples, it is recommended to choose a library preparation kit, sequencing kit, and
platform based on the overall objective of the experiment. In
206
Sandipan Dasgupta and Jeffrey E. Gerst