Quantification-Protocol-miRNA-SCALONMC

Prepare the Qubit® working solution by diluting the Qubit® microRNA reagent 1:200 in Qubit® microRNA buffer as follows:

• 1 µ l of Qubit® microRNA reagent for each sample/standard
• 199 µ l of Qubit® microRNA buffer for each sample/standard

Note: Use a clean plastic tube each time you make Qubit® working solution. Do not mix the working solution in a glass container.

Prepare tube for Standard #1

Label the lid

Add:

• 190 µ l of the Working Solution prepared at STEP 1
• 10 µ l of Qubit® microRNA Standard #1

Mix by vortexing 2–3 seconds, being careful not to create bubbles.

Notes:

• Careful pipetting is critical to ensure that exactly 10 μL of Qubit® microRNA standard #1 is added to 190 μL of Qubit® working solution.

• Use only thin-wall, clear 0.5-mL optical-grade real-time PCR tubes. Acceptable tubes include Qubit® assay tubes (500 tubes, Cat. no. Q32856) or Axygen® PCR-05-C tubes (VWR, part number 10011-830).

Prepare tube for Standard #2

Label the lid

Add:

• 190 µ l of the Working Solution prepared at STEP 1
• 10 µ l of Qubit® microRNA Standard #2

Mix by vortexing 2–3 seconds, being careful not to create bubbles.

Notes:

• Careful pipetting is critical to ensure that exactly 10 μL of Qubit® microRNA standard #2 is added to 190 μL of Qubit® working solution.

• Use only thin-wall, clear 0.5-mL optical-grade real-time PCR tubes. Acceptable tubes include Qubit® assay tubes (500 tubes, Cat. no. Q32856) or Axygen® PCR-05-C tubes (VWR, part number 10011-830).

Prepare the tubes for Samples

Label the lid of each sample as needed

For each sample in each tube, add as follows:

• 198 µ l of the Working Solution prepared at STEP 1
• 2 µ l of sample (purified miRNA)

Mix by vortexing 2–3 seconds, being careful not to create bubbles.

The final volume in each tube should be 200 µ l

Note: Use only thin-wall, clear 0.5-mL optical-grade real-time PCR tubes. Acceptable tubes include Qubit® assay tubes (500 tubes, Cat. no. Q32856) or Axygen® PCR-05-C tubes (VWR, part number 10011-830).

Allow all tubes to incubate at room temperature for 2 minutes.

Proceed to “Read standards and samples”:

On the Home Screen of the Qubit® 2.0 Fluorometer, press microRNA.

The “Standards Screen” will be automatically displayed.

On the Standards screen, press Yes to read the standards.

Insert the tube containing Standard #1 into the sample chamber, close the lid, then press Read.

When the reading is complete (~3 seconds), remove Standard #1.

Insert the tube containing Standard #2 into the sample chamber, close the lid, then press Read.

When the reading is complete, remove Standard #2.

When the calibration is complete, the instrument displays the Sample screen.

Insert a sample tube into the sample chamber, close the lid, then press Read.

When the reading is complete (~3 seconds), remove the sample tube.

Note: The instrument displays the results on the Sample screen in ng/mL.

The value displayed corresponds to the concentration after your sample was diluted into the assay tube.

To find the concentration of the samples as ng/ µ l, use the “Dilution Calculator” feature of the Qubit® 2.0 Fluorometer as follows:

• Press Calculate Stock Conc. The “Dilution Calculator” screen containing the volume roller wheel is displayed.

• Using the volume roller wheel, select the volume of your original sample as 2 µ l. When you stop scrolling, the Qubit® 2.0 Fluorometer calculates the original sample concentration based on the measured assay concentration.

• To change the units in which the original sample concentration is displayed, press ng/mL. A pop-up window showing the current unit selection (as indicated by an adjacent red dash) opens. Select the unit ng/ µ l. To close the unit selection pop-up window, touch anywhere on the screen outside the pop-up.

• To exit the Dilution Calculator screen, press any navigator button on the bottom of the screen or Read Next Sample.

Repeat step 6.5 until all samples have been read.