Real-Time RT-qPCR

2. Bioanalytical Real-Time RT-qPCR Assay Development Protocols

The following section will give you a short history of bioanalytical Real-Time RT-qPCR assay development for siRNA, microRNA (miRNA) DMPK support (pharmacokinetics, pharmacodynamics). When scrolling down through the detailed protocols, please keep in mind the following terminology:
siRNA guide (A) strand: guides the Ago2 endonuclease to the mRNA and hybridizes to it;
siRNA passenger (B) strand: will be degraded
For excellent details on the PK/PD relationship of both strands, please refer to (6).

All the Real-Time RT-qPCR assays published so far for the stem-loop RT primer technology comprise Applied Biosystems kits and devices, at least Alnylam and Merck & Co do.
Most intelligent bioanalytical assay development and validation was performed by Merck & Co scientists by the use of a Ssb-specific siRNA as a model analyte. Ssb is supposed to be ubiquitary expressed among most toxicologic relevant species (Ssb, Sjogren syndrome antigen B). But if you browse the USPTO database for Merck, Sharp & Dohme patent applications, you will find a pretty large siRNA drug development portfolio. Here you go!

 

Applied Biosystems (1): single-stranded microRNA (miRNA) detection
Bioanalytical method: 2-step RT-Real Time qPCR assay
This was the first publication addressing the need for a sensitive quantification method to analyze microRNA expression in tissues. If you deal only with single-stranded small RNAs, the stem-loop RT primer does not have a competitor in the hybridization.

7.5 µl reverse transcription contained:
heat-treated cell lysate w/o RNA purification
50 nM stem-loop RT primer
1x RT Buffer (Applied Biosystems)
0.25 mM dNTPs each
0.25 U/µl RNasin
3.33 U/µl Multiscribe RT (Applied Biosystems)
Program:
30 min 16°C
30 min 42°C
5 min 85°C
hold on 4°C

10 µl real-time qPCR contained:
0.67 µl RT reaction (2 µl were assayed in triplicate)
1x TaqMan® Universal PCR Master Mix
0.2 µM TaqMan® probe
1.5 µM sense primer
0.7 µM antisense primer
Program:
10 min 95°C
15 sec 95°C
1 min 60°C
40 cycles

Academic (2): single-stranded microRNA (miRNA) detection: pulsed RT
Bioanalytical method: 2-step RT-Real Time qPCR assay
Basically as in (1), but with the difference a pulsed reverse transcription is introduced to linear amplify a single-stranded microRNA (miRNA) template. At least I wasn´t able to find any other papers describing a pulsed reverse transcription; also, the authors failed to prove the efficacy of the pulsed RT vs. a conventional RT reaction, though they used less than 30 cycles for qPCR amplification. A single heat shock/ice bath step established the stem-loop RT primer structure.

20 µl reverse transcription contained:
1 µl RNA template (after RNA purification)
50 nM stem-loop RT primer
1x First-strand buffer (Invitrogen)
0.2 mM dNTPs each
0.01 M DTT
0.25 U/µl RNasin
2.5 U/µl SuperScript III RT (Invitrogen)
Program:
1. 30 min 16°C
2. 30 sec 30°C
3. 30 sec 42°C
4. 1 sec 50°C (goto step 2 for another 59 times)
5. 5 min 85°C
hold on 4°C

20 µl real-time qPCR contained:
1 µl RT reaction
1x PCR Buffer (Clontech)
0.2 mM dNTPs each
0.2 µM sense primer
0.2 µM antisense primer
0.4 µl Advantage 2 Polymerase Mix (Clontech)
Program:
2 min 94°C
15 sec 94°C
1 min 60°C
20-40 cycles

Merck & Co (3): double-stranded short interfering RNA (siRNA) detection
Bioanalytical method: 2-step RT-Real Time qPCR assay
This publication was the first describing the quantification in mouse plasma and liver lysate, without the need for RNA purification. Another improvement is the implementation of an universal TaqMan® probe rather than a siRNA-specific, most probably to save costs for probe synthesis and assay optimization.
The explorative RT-qPCR assay development was done with a luciferase-specific siRNA.

Sample preparation:
100 mg/mL tissue lysate in 3M GITC buffer, then diluted 1:50
Assay matrix:
mouse plasma and liver samples
16 µl stem-loop RT primer annealling reaction contained:
4 µl 1:50 diluted siRNA spiked plasma or naïve tissue lysate
12 µl 33.3 nM stem-loop RT primer
Program:
5 min 95°C
2 min 80°C
2 min 70°C
2 min 60°C
2 min 45°C
hold on 4°C

15 µl reverse transcription contained:
8 µl sample/primer mix from above
7 µl TaqMan® MicroRNA RT Kit (Applied Biosystems)*
Program:
30 min 16°C
30 min 42°C
15 min 85°C
hold on 4°C

* which contained:
1x RT Buffer (Applied Biosystems)
0.25 mM dNTPs each
0.25 U/µl RNasin
3.33 U/µl Multiscribe RT (Applied Biosystems)
in accordance to (1) and the kit protocol

20 µl real-time qPCR contained:
5 µl RT reaction
1x TaqMan® Universal PCR Master Mix
0.1 µM TaqMan® probe
1.5 µM sense primer
0.7 µM antisense primer
Program:
10 min 95°C
15 sec 95°C
1 min 60°C
40 cycles
in accordance to (1) and the kit protocol

Academic (4): double-stranded siRNA competitive detection assay
Bioanalytical method: competetive qPCR assay
The authors recognized the need for a one-step real-time assay to eliminate the reverse transcription step. In an ordinary TaqMan® assay various siRNA concentrations were added. The guide (A) strand competes with the sense TaqMan® primer for the hybridization on a DNA template. As a result, the Ct value goes up along with the increasing siRNA guide strand concentration. Interestingly, it was shown that siRNA itself can not prime the taq-mediated polymerization.
The main disadvantage of the competitive qPCR assay is the relative small range of quantification (only 2 orders of magnitude vs. 7 orders of magnitude for the 2-step RT-qPCR assay[1]).

Life Technologies (5): double-stranded short interfering RNA (siRNA) detection
Bioanalytical method: 2-step RT-Real Time qPCR assay
Basically a continuation of the microRNA (miRNA) real-time RT-qPCR assay (1), which was applied to the quantification of double-stranded siRNA. Because the analyte is double-stranded, ABI´s researchers included the initial stem-loop RT primer annealing step as in (3). The real-time RT-qPCR assay itself was performed as described in (1).

Sample preparation:
The mirVana™ PARIS™ Kit was applied to extract total RNA from mouse liver, kidney, lung, and blood; standards were prepared by siRNA spiking to whole organs (not to lysate).

Merck & Co (6): double-stranded short interfering RNA (siRNA) detection
Bioanalytical method: 2-step RT-Real Time qPCR assay

Sample preparation:
20 mg/mL tissue lysate in Trizol buffer homogenized in a bead mill,
then diluted 1:100; standards were prepared by siRNA spiking to organ homogenates (not whole organs).
Assay matrix:
mouse liver, spleen, kidney, lung, muscle, adipose tissue samples

The description of the RT-qPCR assay is only rudimentary pronounced, but the analyte is the same as for the next publication (Ssb, Sjogren syndrome antigen B).

Merck & Co (7): double-stranded short interfering RNA (siRNA) detection
Bioanalytical method: 2-step RT-Real Time qPCR assay
A milestone in siRNA in vivo PK/PD modelling: It´s no surprise a 3.6 mg/kg single I.V. injection showed Ssb mRNA knockdown in the liver even after 24 hours; but strand-specific RT-qPCR assays demonstrated that both the guide and the passenger strands are equally concentrated in the liver, but only the guide strand is loaded into Ago2 (shown by Ago2 immunoprecipitation, see Ref. [7], Figure 4). Great work!

Sample preparation:
cryo-pulverized tissue (Covaris Cryoprep system),
then homogenized in a lysis buffer, supernatants were assayed; standards were prepared by siRNA spiking to organ homogenates (not whole organs).
Assay matrix:
mouse liver, spleen, tissue samples

The description of the RT-qPCR assay is only rudimentary pronounced, but the analyte is the same as for the previous publication (Ssb, Sjogren syndrome antigen B).

Small footnote: When the Merck paper was accepted on 30 August 2010, the following Alnylam publication was already online only for 7 days! So they couldn´t know of each other´s work, could they?

Alnylam Pharmaceuticals (8): double-stranded siRNA detection
Bioanalytical method: 2-step RT-Real Time qPCR assay
This publications meets the double-stranded nature of the siRNA analyte. Tissue lysate is heat-inactivated in the presense of a detergent, immediately cooled, and supernatants are prepared. Centrifugation produces a really huge pellet! Supernatants are heated again to denature double-stranded siRNA, and directly added to the (cooled) reverse transcription mix.
The bioanalytical RT-qPCR assay dynamic range is 4 orders of magnitude (0.7 µg-70 pg per 1 mL plasma or 1g tissue). But one has to keep in mind the range of quantification of such bioanalytical assays depends on: sequence, modification, liposomal formulation, matrix, phase of the moon, etc …

Assay matrix:
rat liver, plasma samples

Sample preparation:
cryo-pulverized tissue (SPEXP Sample Prep Geno Grinder system),
100 mg/mL lysates in 0.25% Triton X-100 in PBS (pre-heated to 95°C)
10 min 95°C
10 min on ice
supernatants were assayed; standards were prepared by siRNA spiking to naïve tissue lysate incubating at 95°C (not to whole organs);
plasma samples were 1:10 diluted, and treated as above

15 µl reverse transcription contained:
10 µl pre-cooled to 4°C, TaqMan® MicroRNA RT Kit mix (Applied Biosystems)*
5 µl pre-heated 10 min 95°C, diluted sample from above
Program:
30 min 16°C
30 min 42°C
5 min 85°C
hold on 4°C

* which contained:
25 mM stem-loop RT primer
1x RT Buffer (Applied Biosystems)
2.5 mmol/µl dNTPs each
20 U/µl RNasin
50 U/µl Multiscribe RT (Applied Biosystems)
in accordance to (1) and the kit protocol;
Certainly the stem-loop RT primer structure first has to be established, a fact the Alnylam staff simply forgot to mention in their paper.

The real-time qPCR contained:
2 µl RT reaction from above
1x TaqMan® Universal PCR Master Mix
0.2 µM TaqMan® probe
1.5 µM sense primer
0.7 µM antisense primer
The description of the qPCR assay is only rudimentary pronounced (no final volume, no cycling conditions published, but maybe in accordance to (1) and the kit protocol. The qPCR asay was performed in an Applied Biosystems 7900HT system, which is ready for 384 well plates, so it´s likely to be the 10 µl final qPCR assay volume which was applied here.

Academic (9): single-stranded microRNA (miRNA) detection: pulsed RT
This publication is basically the same as for (2).

Merck & Co (10): matrix effects on double-stranded siRNA detection
Bioanalytical method: 2-step RT-Real Time qPCR assay
The bioanalytical method is a continuation of the sample prep method described in (3), combined with the real-time RT-qPCR assay published in (6). Because the target for knockdown is again the Ssb mRNA in the liver, thermal conditions for the molecular reactions published in (3) were adopted accordingly.

Some interesting aspects from the bioanalytical assay developer´s point of view:
1. For the first time, the authors compared the RT-qPCR assay performance of crude tissue homogenate vs. mirVana™ miRNA isolates from the same homogenates.
2. Also, Merck & Co scientists compared naked vs. lipoplexed siRNA in the assay.

Assay matrix:
mouse EDTA plasma and liver samples

Sample preparation:
tissue samples pre-served in RNAlater®
100 mg/mL tissue lysate in 3M GITC buffer using a bead mill
EDTA plasma was diluted 1:100 in water containing 0.4 U/µl RNasin

10 µl stem-loop RT primer annealling reaction contained:
5 µl diluted siRNA spiked plasma or naïve tissue lysate
1 µl 10x RT Buffer
4 µl 0.5 µM stem-loop RT primer
Program:
10 min 94°C
2 min 75°C
3 min 60°C
3 min 50°C
3 min 30°C
hold on 4°C

15 µl reverse transcription contained:
10 µl sample/primer mix from above
5 µl TaqMan® MicroRNA RT Kit mix directly added! (Applied Biosystems)*
Program:
30 min 16°C
30 min 42°C
5 min 85°C
hold on 4°C

* which contained:
1x RT Buffer (Applied Biosystems)
25 mM dNTPs each
0.19 µl RNasin from the kit
50 Units Multiscribe RT (Applied Biosystems)
in accordance to (1) and the kit protocol

20 µl real-time qPCR contained:
5 µl RT reaction
1x TaqMan® Universal PCR Master Mix
0.2 µM TaqMan® probe
0.1 µM sense primer
0.1 µM antisense primer

The description of the RT-qPCR assay is only rudimentary pronounced, but the analyte is the same as for the previous publication (Ssb, Sjogren syndrome antigen B).

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