10. Flow cytometer with 488 nm and 560 nm filters for detection
of green and red fluorescence (or as is appropriate for the
fluorophore used).
11. Sorting buffer: 0.5% Bovine serum albumin (BSA), 2 mM
EDTA, in PBS, pH 7.2, degassed and cooled to 4–8
C
before use.
12. Complete culture media (DMEM, supplemented with sodium
pyruvate and penicillin-streptomycin), cooled to 4–8
C
before use.
13. R-phycoerythrin (PE)-anti-mouse CD321 (Jam1) antibody
(BD-Pharmingen).
2.3 RNA Extraction
and Quality Control
1. Commercial RNA extraction kit for extracting up to 100 μg of
total RNA.
2. NanoDrop microvolume spectrophotometer.
3. Agilent TapeStation (for checking RNA integrity).
2.4 Reverse
TranscriptionPolymerase Chain
Reaction
1. 1.7 mL Microcentrifuge tubes.
2. 200 μL PCR tubes.
3. Commercial RNase-free DNase I kit.
4. First-strand cDNA synthesis kit.
5. Commercial PCR mix, consisting of Taq polymerase, reaction
buffers, and dNTP mixture.
6. PCR thermal cycler.
7. Primers to detect the transfer of a validated mRNA: In this
protocol, forward and reverse primers were designed to specifically detect the MS2 aptamer-tagged β-actin mRNA, which is
known to transfer from MBS-MEFs to MCF7 cells and serves
as a positive control for the transfer of RNA (Fig. 1).
8. Molecular biology-grade agarose.
9. SB running buffer: 0.8% NaOH, 4.5% boric acid (H 3 BO 3 ) in
double-distilled water (DDW).
10. Ethidium bromide.
11. Power supply (300 V).
2.5 Deep Sequencing
1. Library preparation kit: The kit is selected with respect to the
experimental design in mind (i.e., detection of the transfer of
mRNA, miRNA, or lncRNA). For example, commercial kits for
the preparation of the different types of RNA for sequencing
are available.
2. Sequencing kit and the platform, selected according to experimental design.
Use of RNA Tagging as a Control for RNA Transferome Analysis
199
of green and red fluorescence (or as is appropriate for the
fluorophore used).
11. Sorting buffer: 0.5% Bovine serum albumin (BSA), 2 mM
EDTA, in PBS, pH 7.2, degassed and cooled to 4–8
C
before use.
12. Complete culture media (DMEM, supplemented with sodium
pyruvate and penicillin-streptomycin), cooled to 4–8
C
before use.
13. R-phycoerythrin (PE)-anti-mouse CD321 (Jam1) antibody
(BD-Pharmingen).
2.3 RNA Extraction
and Quality Control
1. Commercial RNA extraction kit for extracting up to 100 μg of
total RNA.
2. NanoDrop microvolume spectrophotometer.
3. Agilent TapeStation (for checking RNA integrity).
2.4 Reverse
TranscriptionPolymerase Chain
Reaction
1. 1.7 mL Microcentrifuge tubes.
2. 200 μL PCR tubes.
3. Commercial RNase-free DNase I kit.
4. First-strand cDNA synthesis kit.
5. Commercial PCR mix, consisting of Taq polymerase, reaction
buffers, and dNTP mixture.
6. PCR thermal cycler.
7. Primers to detect the transfer of a validated mRNA: In this
protocol, forward and reverse primers were designed to specifically detect the MS2 aptamer-tagged β-actin mRNA, which is
known to transfer from MBS-MEFs to MCF7 cells and serves
as a positive control for the transfer of RNA (Fig. 1).
8. Molecular biology-grade agarose.
9. SB running buffer: 0.8% NaOH, 4.5% boric acid (H 3 BO 3 ) in
double-distilled water (DDW).
10. Ethidium bromide.
11. Power supply (300 V).
2.5 Deep Sequencing
1. Library preparation kit: The kit is selected with respect to the
experimental design in mind (i.e., detection of the transfer of
mRNA, miRNA, or lncRNA). For example, commercial kits for
the preparation of the different types of RNA for sequencing
are available.
2. Sequencing kit and the platform, selected according to experimental design.
Use of RNA Tagging as a Control for RNA Transferome Analysis
199