Table 2 Formulation of the simplex and multiplex RT-PCR reagent mixes.

From: Microdroplet-based one-step RT-PCR for ultrahigh throughput single-cell multiplex gene expression analysis and rare cell detection

Reagents

Unit

Conventional RT-qPCR

Droplet RT-PCR

Simplex (before optimization)

Simplex

Multiplex

IVT multiplex

Crude cell multiplex

TrisHCl, pH 8.0

mM

10

30

30

30

30

KCl

mM

55

55

90

70

70

MgCl2

mM

2

2

2.5

2.5

2.5

SUPERase•In RNase Inhibitor (Invitrogen)

U/μL

1

1

1

1

1

Taq polymerase (produced in Iscove lab)

U/μL

0.1

0.3

0.6

0.6

0.6

HotStart-IT Binding Protein (Thermo Scientific)

ng/μL

510

510

950

950

950

dNTPs

mM

0.4

0.4

0.5

0.5

0.5

Forward & reverse primers (Sigma-Aldrich)

uM

0.3

0.9

0.6

0.6

0.6

TaqMan MGB Probe (Applied Biosystems)

nM

250

350

350

350

350

Nonidet P-40

% vol

0.01

0.01

0.01

0.01

0.14

Pluronic F-68 Non-ionic Surfactant (Gibco)

% vol

0

0

0

0.05

0.2

SuperScript III reverse transcriptase (Invitrogen)

U/μL

1

1

1

1

1

Bovine serum albumin (Roche)

ng/μL

150

450

450

1380

1380

DTT (Invitrogen)

mM

1

1

1

1

1

ROX passive reference dye

nM

50

50

50

0

0

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