RNA extraction

RNeasy® Micro Kit ( Manual )

Preparation

¹ Buffer RLT mixture: Add 10 µL β-mercaptoethanol ( β-ME ) to 1 mL Buffer RLT. The mixture can be stored at room temperature for up to 1 month.

² Buffer RPE working solution: Add 4 volumes of ≥ 96% ethanol to Buffer RPE. In this case, add 2000 µL ethanol to 500 µL Buffer RPE.

³ DNase I stock solution: Dissolve the lyophilized DNase I in 550 µL RNase-free water. To avoid loss of DNase I, do not open the vial. Inject RNase-free water into the vial using an RNase-free needle and syringe. Mix gently by inverting the vial. Do not vortex. Store the stock solution as single use aliquots at -20°C (9 months) or 2-8°C (1 month). Do not refreeze after thawing. Located at 4°C, b cabinet.

⁴ DNase I incubation mix: Add 10 µL DNase I stock solution to 70 µL Buffer RDD, mix by inverting the tube, and put on ice.

Procedure

All instruments should be sterilized prior to the experiment

Step 1: Cell lysis

1. Dissect ≤ 5 mg tissue ( around 30 root tips ) and dab the surface dry gently (better to use KimWipes paper).

• Put the tissue into an eppendorf, slightly centrifuge to spin down the tissue to the bottom of the eppendorf. If the tissue is not used immediately, store the sample at -80°C.

3. Close the eppendorf lid tightly, and then immediately put the eppendorf into liquid nitrogen ( it will float up by itself, no worry ).
4. The drill of the TissueLyser instrument should be pre-cold, by inserting it into the liquid nitrogen.
5. Take out the eppendorf and powderize the tissue with the TissueLyser instrument ( this step is extremely important to yield high quantity and quality of RNA, and this step also should be finished as quick as possible ).
6. Add 350 µL Buffer RLT mixture into the eppendorf and immediately vortex to homogenize the buffer and the powderized tissue ( 10-15 secs )
7. Now we get the raw lysate. Place the eppendorf on ice to inhibit enzymatic activity of the lysate.

Step 2: Collect the lysate supernatant

8. Centrifuge the raw lysate ( 3 mins, 15000 rpm ).
9. Carefully take the supernatant to a new eppendorf, and then place on ice.
10. Add 350 µL 70% EtOH into the sample, mix well with pipetting. DO NOT centrifuge. Proceed to the next step immediately.

Step 3: Wash (1)

11. Transfer the sample ( including any precipitate that may have formed ) to an RNeasy MinElute® spin column in a 2 mL collection tube. Close the lid and centrifuge ( 4°C, 1 min, ≥ 15000 rpm ). Discard the flow-through and reuse the collection tube for the next step.

12. Add 350 µL Buffer RW1 to the RNeasy MinElute® spin column. Close the lid and centrifuge ( 4°C, 1 min, ≥ 15000 rpm ). Discard the flow-through and reuse the collection tube for the next step.

Step 4: Digest DNA

13. Add the DNase I incubation mix (80 µL) to the RNeasy MinElute® spin column membrane. Place on benchtop at room temperature for 15 mins ~ 1 hour.

• Be sure to add the DNase I incubation mix directly to the RNeasy MinElute spin column membrane. DNase digestion will be incomplete if part of the mix sticks to the walls or the O-ring of the spin column
• The DNase I incubation mix (80 µL) should be fresh-prepared. Add 10 µL DNase I stock solution to 70 µL Buffer RDD, mix by inverting the tube, and put on ice. Please refer to the first page for details.

Step 5: Wash (2)

14. Add 350 µL Buffer RW1 to the RNeasy MinElute® spin column. Close the lid and centrifuge ( 4°C, 1 min, ≥ 15000 rpm ). Discard the flow-through and collection tube.

15. Place the RNeasy MinElute® spin column in a new 2 mL collection tube (supplied). Add 500 µL Buffer RPE to the spin column. Close the lid and centrifuge ( 4°C, 1 min, ≥ 15000 rpm ). Discard the flow-through and reuse the collection tube.

16. Add 500 µL 80% EtOH to the spin column. Close the lid and centrifuge 2 times ( 2 × 2 mins, ≥ 15000 rpm ). Discard the flow-through and collection tube.

• After centrifugation, carefully remove the RNeasy MinElute spin column from the collection tube so that the column does not contact the flow-through. Otherwise, carryover of ethanol will occur.

17. Place the RNeasy MinElute® spin column in a new 2 mL collection tube (supplied). Open the lid and centrifuge at full speed ( 4°C, 15 mins, ≥ 15000 rpm ) to dry the membrane. Discard the flow-through and collection tube.

• To avoid damage to their lids, place the spin columns into the centrifuge with at least one empty position between columns. Orient the lids so that they point in a direction opposite to the rotation of the rotor (e.g., if the rotor rotates clockwise, orient the lids counterclockwise)

Step 6: Elute RNA

18. Place the RNeasy MinElute® spin column in a new 1.5 mL RNase-free low-binding collection tube (supplied). Add 14 µL RNase-free water directly to the center of the spin column membrane. Close the lid gently, and centrifuge at full speed ( 4°C, 1 ~ 15 mins, ≥ 15000 rpm ) to elute the RNA.

Step 7: RNA quality & quantity measurement

19. Measure the RNA quality and quantity.
20. Store the RNA at −80°C.

Materials Provided by the kit

Buffer RLT (store at 15 - 25°C)
Buffer RPE (working solution) (store at 15 - 25°C)
Buffer RDD (store at 15 - 25°C)
Buffer RW1 (store at 15 - 25°C)
DNase Set (RNase-Free) (store at 2 - 8°C)
RNeasy MinElute® spin columns (store at 2 - 8°C)
2 mL collection tube
1.5 mL collection tube

• Buffer RLT is a lysis buffer for lysing cells and tissues
• Buffer RPE is a 55 ml concentrated buffer for washing membrane-bound RNA
• Buffer RDD provides efficient on-column digestion of DNA and also ensures that the RNA remains bound to the column
• Buffer RW1 is for washing membrane-bound RNA
• RNeasy MinElute® spin columns ( pink color lid ) stored at 4°C, b cabinet

RNA quantification with Qubit™

Qubit™ RNA HS Assay Kit

Procedure

Materials

For best results, ensure that all materials and reagents are at room temperature.

1. Set up the required number of Qubit™ Assay Tubes for standards and samples. This assay requires 2 standards ( Component C and D ).

• Note: Use only thin-wall, clear, 0.5 mL PCR tubes ( Cat. No. Q32856 ) for the Qubit™ 4 Fluorometer.

2. Label the tube lids.

• Note: Do not label the side of the tubes, that will affect light penetration. Label the lid of each standard tube correctly, as the calibration of the Qubit™ Fluorometer requires the standards to be inserted into the instrument in the right order.

Step 1: Make working solution

The working solution is a mixture of Component A (dye) and Component B (HS buffer), with a ratio of 1 : 199. For example, to make 10 tubes (8 samples + 2 standards), add 10 µL dye into 1990 µL HS buffer.

1. Prepare the working solution by diluting Component A 1:200 in Component B for each tube ( sample + standard solutions ). For example, 10 µL Component A + 1990 µL Component B for 10 tubes ( 8 samples + 2 standards ).

• Note: DO NOT mix the working solution in a glass container. Use clean plastic tube

Step 2: Prepare standard solutions

Step 3: Prepare sample solutions

2. Add the working solution to each tube such that the final volume is 200 µL.

	Standard assay tubes	User sample assay tubes
Volume of working solution	190 µL	180 – 199 µL
Volume of standard	10 µL	—
Volume of user sample	—	1 – 20 µL
Total volume in each assay tube	200 µL	200 µL

5. Add 10 µL of Component C ( standard # 1 ) and D ( standard # 2 ) separately to the appropriate tubes.
6. Add 1 µL of each user sample to the appropriate tubes.
7. Vigorously vortex for 3 ~ 5 seconds.
8. Incubate at room temperature for 2 minutes. Then proceed to read standards and samples with the Qubit™ 4 Fluorometer.

Reading standards and samples

1. On the Home screen, touch RNA, then select RNA High Sensitivity as the assay type. Touch Read standards to proceed.
2. Insert the tube containing Standard # 1 into the sample chamber, close the lid, then touch Read standard. When the reading is comnplete, remove Standard # 1.
3. Insert the tube containing Standard # 2 into the sample chamber, close the lid, then touch Read standard. When the reading is comnplete, remove Standard # 2.
4. Touch Run samples.
5. On the assay screen, select the Sample volume and Unit.

• Touch the + or - buttons on the wheel, or anywhere on the wheel itself, to select the sample volume added to the assay tube ( 1 µL in this protocol ).
• From the Unit dropdown menu, select the units for the output sample concentration.

6. Insert a sample tube into the sample chamber, close the lid, then touch Read tube. When the reading is complete ( ~ 3 seconds ), remove the sample tube.

• The top value ( in large font ) is the concentration of the original sample and the bottom value is the dilution concentration.

7. Repeat step 6 until all samples have been read.

Materials Provided by the kit

^* Q32852: Catalogue number. When stored as directed, kits are stable for 6 months.

^☨ Component A: Stored at 2 ~ 8°C ( short-term storage ) or ≤ -20°C ( long-term storage ), desiccated, protect from light. Located at -80°C, upper door, 2^nd layer, 2^nd column, 3^rd drawer, 10 µL pale blue aliquots in PCR tubes.

^§ Component B: Stored at ≤ 30 °C. Located at VI.

^※ Component C & D: Stored at 2 ~ 8°C ( short-term storage ) or ≤ -20°C ( long-term storage ). Located at -80°C, upper door, 2^nd layer, 2^nd column, 3^rd drawer.

Materials NOT Provided by the kit

1. Plastic container ( RNase-free, disposable ) for mixing the Qubit™ working solution;
2. Qubit™ Assay Tubes ( 500 tubes, Cat. No. Q32856 );