TITLE: Quick Protocol for Monarch® Total RNA Miniprep Kit (NEB #T2010)
AUTHORS: New  England   Biolabs

[DESCRIPTION]
Quick Protocol for Monarch® Total RNA Miniprep Kit (NEB #T2010).
 
Quickly and easily purify up to 100 µg of high-quality total RNA from multiple sample types – all with one kit!
For use with blood, cells and tissues
Also works with tough to lyse samples (bacteria, yeast, plant)
Effectively purifies total RNA of all sizes, including small RNAs >20 nt
Efficient genomic DNA removal (column and DNase I-based)
Contains Proteinase K for processing of tissues and blood samples
Includes RNA Protection Reagent for sample preservation
Excellent value
Kit components available separately

[BEFORE_START]
Addition of RNA Lysis Buffer and all subsequent steps should be performed at room temperature to prevent formation of precipitate. If samples are accidentally placed on ice and precipitate forms, allow the samples to return to room temperature to resolubilize before loading onto the column.
Protocols are also available for RNA reaction cleanup, RNA fractionation, and extraction of RNA from other sample types (including those in preservation reagents or TRIzol®).
 
For the 50 prep kit:
Reconstitute DNase I by adding 275 µL. Gently invert to mix. Aliquot for storage at -20 °C to minimize freeze-thaw cycles.
Reconstitute Proteinase K (Prot K) by adding 1040 µL. Vortex and store at -20 °C.
Add 100 mL to the 25 mL.

[GUIDELINES]
We recommend that first-time users of this kit review the product manual before starting. The manual provides additional relevant information to consider at various steps. This quick protocol is meant for experienced users.
 
RNA Purification Consists of Two Stages:
PART 1: Sample Disruption and Homogenization (Differs depending on starting material)
PART 2: RNA Binding and Elution
 
Have any questions?
Our tech support scientists would be happy to help. Email us at info@neb.com.

[STEPS]
SECTION: PART 1: Sample Disruption and Homogenization
1. Please select your starting material of the following:
- Cultured Mammalian Cells
- Mammalian Whole Blood (Fresh or Frozen)
- Tissue or Leukocytes
- Tough-to-Lyse Samples (bacteria, yeast, plant, etc.) using Mechanical Lysis
SECTION: PART 1: Sample Disruption and Homogenization
2. Pellet cells by centrifugation 500 x g, 1 min.
SECTION: PART 1: Sample Disruption and Homogenization
3. Discard supernatant.
SECTION: PART 1: Sample Disruption and Homogenization
4. Resuspend pellet in RNA Lysis Buffer (according to table below) by pipetting gently to avoid foaming. 
 
   AMOUNT OF CELLS VOLUME OF RNA LYSIS BUFFER   up to 3 x 106 300 µl   3 x 106 to 1 x 107  ≥ 600 µl   
 
SECTION: PART 2: RNA BINDING AND ELUTION
5. Transfer up to 800 µL of the sample from PART 1 to a gDNA removal column (light blue) fitted with a collection tube.

For sample identification, label collection tubes, as gDNA removal columns will be discarded after spinning.
 
 

SECTION: PART 2: RNA BINDING AND ELUTION
6. Spin at 16000 x g for 30 s to remove most of the gDNA. SAVE THE FLOW-THROUGH (RNA partitions here). Discard the gDNA removal column.

SECTION: PART 2: RNA BINDING AND ELUTION
7. Add an equal volume of ethanol (≥ 95 %) to the flow-through and mix thoroughly by pipetting. Do not vortex. To exclude RNA ≷ 200 nt, add only 1/2 volume ethanol to flow-through.
SECTION: PART 2: RNA BINDING AND ELUTION
8. Transfer mixture to an RNA purification column (dark blue) fitted with a collection tube. 

Spin at 16000 x g for 30 s. Discard flow-through. If further gDNA removal is essential for downstream applications, proceed to on-column DNase I treatment, steps 9-11 (recommended). If not, proceed to Step 12.

SECTION: PART 2: RNA BINDING AND ELUTION
9. [Optional (but recommended)] On-column DNase I treatment for enzymatic removal of residual gDNA:
Add 500 µL and spin at 16000 x g for 30 s. Discard flow-through.

SECTION: PART 2: RNA BINDING AND ELUTION
10. [Optional (but recommended)] In an RNase-free microfuge tube (not included), combine 5 µL with 75 µL and pipet directly to the top of the column matrix.
SECTION: PART 2: RNA BINDING AND ELUTION
11. [Optional (but recommended)] Incubate for 15 min at Room temperature.
SECTION: PART 2: RNA BINDING AND ELUTION
12. Add 500 µL and spin at 16000 x g for 30 s. Discard flow-through.
SECTION: PART 2: RNA BINDING AND ELUTION
13. Add 500 µL and spin at 16000 x g for 30 s. Discard flow-through.
SECTION: PART 2: RNA BINDING AND ELUTION
14. Add another 500 µL and spin at 16000 x g for 2 min. 
SECTION: PART 2: RNA BINDING AND ELUTION
15. Transfer column to an RNase-free microfuge tube (not included). Use care to ensure the tip of the column does not contact the flow-through. If in doubt, re-spin at 16000 x g for 1 min to ensure no ethanol is carried over.

SECTION: PART 2: RNA BINDING AND ELUTION
16. Add 30 µL-100 µL directly to the center of column matrix and spin at 16000 x g for 30 s.
 

SECTION: PART 2: RNA BINDING AND ELUTION
17. Place RNA on ice if being used for downstream steps, at -20 °C for short-term storage (less than 1 week), or at -80 °C for long-term storage. Addition of EDTA to 0.1 Milimolar (mM)-1.0 Milimolar (mM) may reduce the activity of any contaminating RNases.
SECTION: PART 1: Sample Disruption and Homogenization
2. Add an equal volume (up to 200 µL) of DNA/RNA Protection Reagent (2X concentrate) to an aliquot of whole blood and vortex briefly. 
 

SECTION: PART 1: Sample Disruption and Homogenization
3. For every 400 µl of DNA/RNA Protection Reagent/blood mixture, add 10 µL. Vortex briefly and incubate at Room temperature for 30 min.
SECTION: PART 1: Sample Disruption and Homogenization
4. Add an equal volume of isopropanol (not included) and vortex briefly.
SECTION: PART 2: RNA BINDING AND ELUTION
5. Transfer mixture to an RNA purification column (dark blue) fitted with a collection tube. 

Spin at 16000 x g for 30 s. Discard flow-through. If further gDNA removal is essential for downstream applications, proceed to on-column DNase I treatment, steps 6-8 (recommended). If not, proceed to Step 9. 
SECTION: PART 2: RNA BINDING AND ELUTION
6. [Optional (but recommended)] On-column DNase I treatment for enzymatic removal of residual gDNA:
Add 500 µL and spin at 16000 x g for 30 s. Discard flow-through.
SECTION: PART 2: RNA BINDING AND ELUTION
7. [Optional (but recommended)] In an RNase-free microfuge tube (not included), combine 5 µL with 75 µL and pipet directly to the top of the column matrix.
SECTION: PART 2: RNA BINDING AND ELUTION
8. [Optional (but recommended)] Incubate for 15 min at Room temperature.
SECTION: PART 2: RNA BINDING AND ELUTION
9. Add 500 µL and spin at 16000 x g for 30 s. Discard flow-through.
SECTION: PART 2: RNA BINDING AND ELUTION
11. Add another 500 µL and spin at 16000 x g for 2 min. 
SECTION: PART 2: RNA BINDING AND ELUTION
12. Transfer column to an RNase-free microfuge tube (not included). Use care to ensure the tip of the column does not contact the flow-through. If in doubt, re-spin at 16000 x g for 1 min to ensure no ethanol is carried over.
SECTION: PART 2: RNA BINDING AND ELUTION
13. Add 30 µL-100 µL directly to the center of column matrix and spin at 16000 x g for 30 s.
 


SECTION: PART 2: RNA BINDING AND ELUTION
14. Place RNA on ice if being used for downstream steps, at -20 °C for short-term storage (less than 1 week), or at -80 °C for long-term storage. Addition of EDTA to 0.1 Milimolar (mM)-1.0 Milimolar (mM) may reduce the activity of any contaminating RNases.
SECTION: PART 1: Sample Disruption and Homogenization
2. Determine the amount of 1 X that you will need according to the table below. Prepare the 1X aliquot by diluting the 2X stock with nuclease-free water (not included for this step).
 
   Sample Input Amount Volume 1X DNA/RNA Protection Reagent   Tissue (up to 10 mg) 300 µl   Tissue (10–30 mg) 300–600 µl   Tissue (30–50 mg) ≥ 600 µl   Leukocytes (up to 3 x 106) 300 µl   Leukocytes (3 x 106 to 1 x 107) ≥ 600 µl   
SECTION: PART 1: Sample Disruption and Homogenization
3. Add 1 X to sample.
 
SECTION: PART 1: Sample Disruption and Homogenization
4. For every 300 µl of DNA/RNA Protection Reagent/sample mixture, add 30 µL + 15 µL. Vortex briefly and incubate according to the chart below:
 
   Sample Type Incubation Time at 55°C**   Homogenized Tissues 5 min   Solid Tissues 5-30 min   Leukocytes 30 min   
* Doubling the Prot K volume may increase RNA yield for some tissues.
** Longer incubation times may result in decreased RNA integrity. Optimal time should be determined empirically, as yield and integrity vary with incubation time.
SECTION: PART 1: Sample Disruption and Homogenization
5. Vortex sample briefly and spin for 2 min (16000 x g) to pellet debris.
SECTION: PART 1: Sample Disruption and Homogenization
6. Transfer supernatant to an RNase-free microfuge tube (not included).
SECTION: PART 1: Sample Disruption and Homogenization
7. Add an equal volume of RNA Lysis Buffer and vortex briefly.
SECTION: PART 2: RNA BINDING AND ELUTION
8. Transfer up to 800 µL of the sample from PART 1 to a gDNA removal column (light blue) fitted with a collection tube.

For sample identification, label collection tubes, as gDNA removal columns will be discarded after spinning.
 
 
SECTION: PART 2: RNA BINDING AND ELUTION
9. Spin at 16000 x g for 30 s to remove most of the gDNA. SAVE THE FLOW-THROUGH (RNA partitions here). Discard the gDNA removal column.

SECTION: PART 2: RNA BINDING AND ELUTION
10. Add an equal volume of ethanol (≥ 95 %) to the flow-through and mix thoroughly by pipetting. Do not vortex. To exclude RNA ≷ 200 nt, add only 1/2 volume ethanol to flow-through.
SECTION: PART 2: RNA BINDING AND ELUTION
11. Transfer mixture to an RNA purification column (dark blue) fitted with a collection tube. 

Spin at 16000 x g for 30 s. Discard flow-through. If further gDNA removal is essential for downstream applications, proceed to on-column DNase I treatment, steps 12-14 (recommended). If not, proceed to Step 15.
SECTION: PART 2: RNA BINDING AND ELUTION
12. [Optional (but recommended)] On-column DNase I treatment for enzymatic removal of residual gDNA:
Add 500 µL and spin at 16000 x g for 30 s. Discard flow-through.
SECTION: PART 2: RNA BINDING AND ELUTION
13. [Optional (but recommended)] In an RNase-free microfuge tube (not included), combine 5 µL with 75 µL and pipet directly to the top of the column matrix.
SECTION: PART 2: RNA BINDING AND ELUTION
14. [Optional (but recommended)] Incubate for 15 min at Room temperature.
SECTION: PART 2: RNA BINDING AND ELUTION
15. Add 500 µL and spin at 16000 x g for 30 s. Discard flow-through.
SECTION: PART 2: RNA BINDING AND ELUTION
16. Add 500 µL and spin at 16000 x g for 30 s. Discard flow-through.
SECTION: PART 2: RNA BINDING AND ELUTION
17. Add another 500 µL and spin at 16000 x g for 2 min. 
SECTION: PART 2: RNA BINDING AND ELUTION
18. Transfer column to an RNase-free microfuge tube (not included). Use care to ensure the tip of the column does not contact the flow-through. If in doubt, re-spin at 16000 x g for 1 min to ensure no ethanol is carried over.
SECTION: PART 2: RNA BINDING AND ELUTION
19. Add 30 µL-100 µL directly to the center of column matrix and spin at 16000 x g for 30 s.
 

SECTION: PART 2: RNA BINDING AND ELUTION
20. Place RNA on ice if being used for downstream steps, at -20 °C for short-term storage (less than 1 week), or at -80 °C for long-term storage. Addition of EDTA to 0.1 Milimolar (mM)-1.0 Milimolar (mM) may reduce the activity of any contaminating RNases.
SECTION: PART 1: Sample Disruption and Homogenization
2. 
 

Determine the amount of 1 X that you will need according to the table below. Prepare the 1X aliquot by diluting the 2X stock with nuclease-free water (not included for this step).
 
   Sample Input Amount Volume 1X DNA/RNA Protection Reagent   Bacteria  Yeast Plant     ≤ 5x107 ≤ 5x106   400 µl   ≤ 5x107-1x109 ≤ 5x106-5x107 ≤ 100 mg 800 µl   
SECTION: PART 1: Sample Disruption and Homogenization
3. Add 1 X to sample.
SECTION: PART 1: Sample Disruption and Homogenization
4. Disrupt sample mechanically using a bead mill or similar device.
SECTION: PART 1: Sample Disruption and Homogenization
5. Transfer lysate/homogenate to an RNase-free microfuge tube (not included).
SECTION: PART 1: Sample Disruption and Homogenization
6. Spin for 2 min (16000 x g) to pellet debris.
SECTION: PART 1: Sample Disruption and Homogenization
7. Transfer supernatant to an RNase-free microfuge tube (not included).
SECTION: PART 1: Sample Disruption and Homogenization
8. Add an equal volume of RNA Lysis Buffer and vortex briefly.
SECTION: PART 2: RNA BINDING AND ELUTION
9. Transfer up to 800 µL of the sample from PART 1 to a gDNA removal column (light blue) fitted with a collection tube.
For sample identification, label collection tubes, as gDNA removal columns will be discarded after spinning.
 
 
SECTION: PART 2: RNA BINDING AND ELUTION
10. Spin at 16000 x g for 30 s to remove most of the gDNA. SAVE THE FLOW-THROUGH (RNA partitions here). Discard the gDNA removal column.
SECTION: PART 2: RNA BINDING AND ELUTION
11. Add an equal volume of ethanol (≥ 95 %) to the flow-through and mix thoroughly by pipetting. Do not vortex. To exclude RNA ≷ 200 nt, add only 1/2 volume ethanol to flow-through.
SECTION: PART 2: RNA BINDING AND ELUTION
12. Transfer mixture to an RNA purification column (dark blue) fitted with a collection tube. 

Spin at 16000 x g for 30 s. Discard flow-through. If further gDNA removal is essential for downstream applications, proceed to on-column DNase I treatment, steps 13-15 (recommended). If not, proceed to Step 16.
SECTION: PART 2: RNA BINDING AND ELUTION
13. [Optional (but recommended)] On-column DNase I treatment for enzymatic removal of residual gDNA:
Add 500 µL and spin at 16000 x g for 30 s. Discard flow-through.
SECTION: PART 2: RNA BINDING AND ELUTION
14. [Optional (but recommended)] In an RNase-free microfuge tube (not included), combine 5 µL with 75 µL and pipet directly to the top of the column matrix.
SECTION: PART 2: RNA BINDING AND ELUTION
15. [Optional (but recommended)] Incubate for 15 min at Room temperature.
SECTION: PART 2: RNA BINDING AND ELUTION
16. Add 500 µL and spin at 16000 x g for 30 s. Discard flow-through.
SECTION: PART 2: RNA BINDING AND ELUTION
18. Add another 500 µL and spin at 16000 x g for 2 min. 
SECTION: PART 2: RNA BINDING AND ELUTION
19. Transfer column to an RNase-free microfuge tube (not included). Use care to ensure the tip of the column does not contact the flow-through. If in doubt, re-spin at 16000 x g for 1 min to ensure no ethanol is carried over.
SECTION: PART 2: RNA BINDING AND ELUTION
20. Add 30 µL-100 µL directly to the center of column matrix and spin at 16000 x g for 30 s.
 
SECTION: PART 2: RNA BINDING AND ELUTION
21. Place RNA on ice if being used for downstream steps, at -20 °C for short-term storage (less than 1 week), or at -80 °C for long-term storage. Addition of EDTA to 0.1 Milimolar (mM)-1.0 Milimolar (mM) may reduce the activity of any contaminating RNases.
SECTION: PART 2: RNA BINDING AND ELUTION
10. Add 500 µL and spin at 16000 x g for 30 s. Discard flow-through.
SECTION: PART 2: RNA BINDING AND ELUTION
17. Add 500 µL and spin at 16000 x g for 30 s. Discard flow-through.