TITLE: RNA EXTRACTION FROM WHEAT STIGMAS STORED IN DNA/RNA SHIELD
AUTHORS: Marina Millán Blánquez  

[DESCRIPTION]
Despite the importance of understanding the molecular processes plants go through under field conditions, transcriptomic studies are often difficult to perform on field samples since access to liquid nitrogen is not always possible. Here, I present a protocol optimised for the extraction of high-quality RNA from wheat stigmas stored in DNA/RNA shield as a substitute to liquid nitrogen. This protocol is based on a first step of TRIzol/Chloroform phase separation and a second step to purify the aqueous phase using RNA Clean and Concentrator kit from ZYMO-Research. The addition of in-tube DNase treatment during the purification step makes this protocol also ideal for tissues with high carbohydrate content, like the wheat stigma, as DNA tends to co-precipitate with the carbohydrates and consequently overriding the on-column purification steps. This protocol has been tested in other plant tissues such as maize and wheat pollen and floral meristems in wheat with good results.

[BEFORE_START]
RNA is not as stable as DNA and is susceptible to degradation by heat, RNases, and other enzymes.

Always wear gloves, and whenever possible, work quickly to reduce RNA degradation and work under fume hood when using TRIzol and chloroform. 

Keep work area, equipment, and reagents RNase-free (RNase decontamination solution, such as RNaseZap® or RNase AWAY®, may be used).


[STEPS]
SECTION: Phase separation
1. Add two steel ball bearings into each Eppendorf tube containing the samples preserved in DNA/RNA Shield solution (~200 μL should be enough for five stigmas at Waddington stage 10 – i.e., stigmatic branches spreading outwards) (Fig. 1)

  

SECTION: Phase separation
2. Grind stigmas in DNA/RNA Shield solution for 2 mins at full speed in a tissue homogenizer like Geno Grinder. If foam is formed on the surface spin the Eppendorf tubes at the maximum speed for approx. 1 min in the centrifuge.
SECTION: Phase separation
3. Add 800μL of TRIzol reagent (kept at 4 oC). 
SECTION: Phase separation
4. Mix samples by gentle inversion or rocking motion for 2-3 minutes at RT and incubate the lysate at RT for 5 min to allow complete dissociation of nucleoprotein complexes. 
SECTION: Phase separation
5. Centrifuge at 15,000 rpm (no more than 12,000xg) for 10 mins at 4 oC.
SECTION: Phase separation
6. Transfer the supernatant into a fresh 1.5 mL tube. Add 0.2 volumes of chloroform per volume of TRIzol (e.g., 160 μL per 800 μL of TRIzol). Shake the tube vigorously by hand for 15 s. 
SECTION: Phase separation
7. Incubate for 2-3 min at RT.
SECTION: Phase separation
8. Centrifugate at 15,000 rpm (no more than 12,000xg) for 10 mins at 4 oC.
SECTION: The following steps are part of the RNA Clean and Concentrator kit (ZYMO-Research):
9. From now onwards, perform all steps at room temperature and centrifugation at 10,000 – 16,000 x g for 30 seconds

Following TRIzol/Chloroform extraction, carefully transfer the upper aqueous phase into a nuclease-free tube (not provided).
SECTION: The following steps are part of the RNA Clean and Concentrator kit (ZYMO-Research):
10. For each volume of the aqueous phase (as measured or estimated), add 1 volume ethanol (95-100%) and mix.
SECTION: The following steps are part of the RNA Clean and Concentrator kit (ZYMO-Research):
11. Transfer the sample to the Zymo-Spin™ IC Column in a Collection Tube and centrifuge. Discard the flow-through. To process samples >800 μl, Zymo-Spin™ columns may be reloaded.

SECTION: The following steps are part of the RNA Clean and Concentrator kit (ZYMO-Research):
11.1. In-column DNase I treatment:
-Following the RNA binding step (step 11), prewash the column with 400 μl RNA Wash Buffer. Centrifuge and discard the flow-through.
-For each sample to be treated, prepare DNase I reaction mix in an RNase-free tube (not provided). Mix well by gentle inversion:

DNase I                                   5 μl
DNA Digestion Buffer        35 μl

-Add 40 μl reaction mix directly to the column matrix. Incubate at room temperature (20-30ºC) for 15 minutes. Then continue with the RNA purification protocol (step 12).
SECTION: The following steps are part of the RNA Clean and Concentrator kit (ZYMO-Research):
12. Add 400 μl RNA Prep Buffer to the column and centrifuge. Discard the flow-through.
SECTION: The following steps are part of the RNA Clean and Concentrator kit (ZYMO-Research):
13. Add 700 μl RNA Wash Buffer to the column and centrifuge. Discard the flow-through.
SECTION: The following steps are part of the RNA Clean and Concentrator kit (ZYMO-Research):
14. Add 400 μl RNA Wash Buffer to the column and centrifuge for 1 minute ensure complete removal of the wash buffer. Carefully, transfer the column into a nuclease-free tube (not provided).
SECTION: The following steps are part of the RNA Clean and Concentrator kit (ZYMO-Research):
15. Add 15 μl DNase/RNase-Free Water directly to the column matrix and centrifuge.
Alternatively, for highly concentrated RNA use ≥6 μl elution.
SECTION: The following steps are part of the RNA Clean and Concentrator kit (ZYMO-Research):
16. The eluted RNA can be used immediately or stored at -80 °C to prevent degradation (avoid multiple freeze-thaw cycles).
SECTION: RNA quantity and quality
17. To quantify and assess the quality of your RNA sample(s) you can use a spectrophotometer (such as a Nanodrop), agarose gel, or bioanalyzer.