Agilent TapeStation vs. Qubit: RNA concentration.

[evobio]

SCENARIO:

After RNA extraction (RNA-seq is my aim), I check the concentration of the extracted RNA with Qubit, and then run the extracted RNA on Agilent TapeStation 2200 with HSRNA ScreenTape. The concentrations are listed below and the RINe values from the TapeStation are shown in the picture.

Qubit concentrations vs. TapeStation concentrations:
A1 = 14.9 ng/uL vs. 40.6 ng/uL
B1 = 19.4 ng/uL vs. 60.2 ng/uL
C1 = 24.4 ng/uL vs. 135 ng/uL
D1 = 21.5 ng/uL vs. 110 ng/uL
E1 = 32.3 ng/uL vs. ?

QUESTIONS:

1. Which concentration values should I trust: Qubit or TapeStation?

2. Should I trust the RINe values of the TapeStation, even though the error message says "RNA concentration outside the recommended range for RINe", or should I dilute the sample according to the concentration given by the TapeStation and run them again?

3. I've been told to use samples with approximately the same concentration on the TapeStation because the bad concentration of one sample (either too high or too low) can affect the results of the other samples. Is that true? How can one sample affect the result of another?

[pmiguel]

Originally Posted by evobio

SCENARIO:

After RNA extraction (RNA-seq is my aim), I check the concentration of the extracted RNA with Qubit, and then run the extracted RNA on Agilent TapeStation 2200 with HSRNA ScreenTape. The concentrations are listed below and the RINe values from the TapeStation are shown in the picture.

Qubit concentrations vs. TapeStation concentrations:
A1 = 14.9 ng/uL vs. 40.6 ng/uL
B1 = 19.4 ng/uL vs. 60.2 ng/uL
C1 = 24.4 ng/uL vs. 135 ng/uL
D1 = 21.5 ng/uL vs. 110 ng/uL
E1 = 32.3 ng/uL vs. ?

QUESTIONS:

1. Which concentration values should I trust: Qubit or TapeStation?

Probably Qubit. But which dye were you using? As long as you were using the RNA-specific dye, then trust the Qubit.

Originally Posted by evobio

2. Should I trust the RINe values of the TapeStation, even though the error message says "RNA concentration outside the recommended range for RINe", or should I dilute the sample according to the concentration given by the TapeStation and run them again?

I don't know. Do you want to for some reason? What will you do with the RIN score?

Originally Posted by evobio

3. I've been told to use samples with approximately the same concentration on the TapeStation because the bad concentration of one sample (either too high or too low) can affect the results of the other samples. Is that true? How can one sample affect the result of another?

Seems crazy to me, but there are crazy aspects to the Agilent Bioanalyzer. So maybe there are also goofy issues with the TapeStation as well.

--
Phillip

[evobio]

1. I am using the appropriate Qubit dye (Qubit RNA HS Assay Kit).

2. The extracted RNA will be used in the preparation of RNA-seq libraries, so I'm using the RINe scores from the TapeStation as a proxy of the quality of the RNA. So I guess I need to run the samples again with the new concentrations in order to trust completely the RINe scores of the TapeStation.

3. To me that doesn't make sense, because that means one bad sample can give you bad scores for all other samples (even if they are good). If that really is the case, each run should have only one sample, which is absurd.

[pmiguel]

2. Okay.
3. Actually, Agilent has decent tech support. Maybe give them a call about this issue. See if it is true...

--
Phillip