TITLE: Protocol for RNA Extraction from Peanut Samples Using Direct-zol™ RNA Miniprep
AUTHORS: Gabriela Gabriela Paredes

[DESCRIPTION]
RNA extraction is a fundamental step in molecular biology, crucial for downstream applications such as qPCR, sequencing, and gene expression analysis. The process of RNA extraction involves the isolation of RNA from cells or tissues, which must be done in a way that preserves the integrity and purity of the RNA. This is especially important because RNA is highly susceptible to degradation by RNases, which are ubiquitous in the environment.
The extraction process typically involves several key steps: cell lysis, removal of proteins and other contaminants, and purification of the RNA. The Direct-zol‱ RNA Miniprep kit simplifies these steps by allowing for the direct processing of samples in TRIzol‱ or similar reagents, which are known for their strong denaturing properties that inactivate RNases and other degradative enzymes. TRIzol‱ is particularly advantageous for samples with high oil content, such as peanut seeds, as it effectively disrupts the cell membranes and separates nucleic acids from lipids and other cellular components without the need for chloroform, phase separation, or precipitation steps.
Using TRIzol‱ in combination with the Direct-zol‱ RNA Miniprep kit ensures that high-quality total RNA, including small RNAs, can be efficiently extracted even from challenging samples like peanut tissues. This streamlined method reduces the complexity and time required for RNA extraction, making it an ideal choice for researchers working with oil-rich seeds.

[BEFORE_START]
Before starting the RNA extraction, ensure that all materials and reagents are prepared and that you have completed the following steps:
Buffer Preparation: Add ethanol to the Direct-zol‱ RNA PreWash and RNA Wash Buffer concentrates as per the kit instructions.
DNase I Reconstitution: Reconstitute DNase I with DNase/RNase-Free Water and store aliquots as per the kit instructions.
Equipment Check: Verify that the bead beater, centrifuge, and other equipment are functioning properly and set to the appropriate settings.

[GUIDELINES]
Aseptic Technique: Ensure that all steps are performed using aseptic techniques to prevent contamination of RNA samples. Wear gloves, and work in a clean environment, ideally under a laminar flow hood.
Sample Homogenization: Properly homogenize peanut samples using a bead beater to ensure complete cell lysis. Inadequate homogenization can lead to poor RNA yield and contamination.
Ethanol Purity: Use high-purity ethanol (95-100%) in all steps requiring ethanol addition. Lower purity ethanol can lead to inefficient RNA precipitation and lower yields.
Temperature Control: Maintain samples at room temperature during processing. Cold temperatures can cause unwanted precipitation, while high temperatures may degrade RNA.

[STEPS]
SECTION: Preparation Before Starting
2. Sample Preparation:
Tough-to-lyse samples (e.g., peanut tissues): For peanut tissues, which are challenging due to their high oil content, homogenize the samples using a bead beater. Use a sufficient amount of TRIzol‱ or TRI Reagent‱ (at least 800 µl) to ensure complete lysis.
SECTION: Preparation Before Starting
1. Buffer Preparation:
Add ethanol to the Direct-zol™ RNA PreWash concentrate and RNA Wash Buffer concentrate according to the kit instructions.
Reconstitute the lyophilized DNase I with DNase/RNase-Free Water as per the instructions provided in the kit.
SECTION: Preparation Before Starting
3. Bead Beater Instructions:
Use 2.0 mm beads for effective disruption of peanut tissues.
Add the tissue sample and beads to a suitable tube, then add TRIzol®.
Homogenize the sample at high speed for 30-60 seconds. Allow the sample to cool for 1-2 minutes, then repeat the homogenization for another 30-60 seconds.
If the lysate appears viscous or if there is visible precipitation, add additional TRIzol® (100-200 µl) and continue homogenizing until the lysate is smooth and homogeneous.

After homogenization, centrifuge the sample to remove particulate debris and transfer the supernatant to a new RNase-free tube.
SECTION: What to Do if Lysis is Incomplete or Precipitation is Observed
4. Incomplete Lysis:
Ensure that the sample is thoroughly homogenized by increasing the homogenization time or repeating the bead beating cycle.
If the sample remains viscous, indicating incomplete lysis, add additional TRIzol® (100-200 µl) and homogenize again.
Use a larger volume of TRIzol® initially to ensure complete lysis, especially for samples with high lipid content like peanuts.
SECTION: What to Do if Lysis is Incomplete or Precipitation is Observed
5. Precipitation:
Precipitation can occur if the sample is not fully homogenized or if the TRIzol® is not sufficient to dissolve all components.
To resolve this, add more TRIzol® (up to double the original volume) and re-homogenize the sample.
Ensure that the sample is kept at room temperature during processing to prevent premature precipitation of nucleic acids.
SECTION: RNA Extraction Protocol
8. DNase I Treatment (Optional, Recommended):
Add 400 µl of RNA Wash Buffer to the column and centrifuge.
In a separate RNase-free tube, prepare the DNase I mix by adding 5 µl of DNase I (6 U/µl) to 75 µl of DNA Digestion Buffer. Mix gently.
Add the DNase I mix directly to the column matrix. Incubate at room temperature (20-30°C) for 15 minutes.
SECTION: RNA Extraction Protocol
6. Lysis and Binding:
Add an equal volume of ethanol (95-100%) to the homogenized sample in TRIzol® or TRI Reagent®. Mix thoroughly by vortexing.

Example: For 400 µl of the sample, add 400 µl of ethanol.
SECTION: RNA Extraction Protocol
7. RNA Binding:
Transfer the mixture into a Zymo-Spin™ IICR Column in a collection tube. Centrifuge at 10,000-16,000 x g for 30 seconds.

Discard the flow-through and place the column in a new collection tube.
SECTION: RNA Extraction Protocol
10. RNA Elution:
Carefully transfer the column to a new RNase-free tube.
Add 50 µl of DNase/RNase-Free Water directly to the column matrix and centrifuge to elute the RNA.
For a more concentrated RNA sample, use a lower elution volume (≥ 25 µl).
SECTION: RNA Extraction Protocol
9. Column Washing:
After the incubation, add 400 µl of Direct-zol™ RNA PreWash to the column and centrifuge. Discard the flow-through.

Repeat the wash step with another 400 µl of Direct-zol™ RNA PreWash.

Add 700 µl of RNA Wash Buffer to the column and centrifuge for 1 minute to ensure the complete removal of any residual buffer.
SECTION: Post-Extraction Steps
11. Storage: The eluted RNA can be used immediately for downstream applications or stored at -80°C for long-term storage.
Quality Check: Assess RNA quality and quantity using a spectrophotometer (A260/A280 & A260/A230 ratios) and/or agarose gel electrophoresis.