eLabNotebook - Promega's MagneSil* Total RNA mini-Isolation System on the Biomek® FX

eLabNotebook > Nucleic Acid Prep & Purification > RNA Purification > Promega MagneSil*
Total RNA mini-Isolation Biomek® FX

Promega's MagneSil* Total RNA mini-Isolation System on the Biomek® FX

Promega Corporation – www.promega.com

Before You Begin

A. Automated RNA Purification on the Biomek® FX Laboratory Workstation

Figure 1. Overview of the MagneSil* Total RNA mini-Isolation System protocol.

B. Initial Deck Configuration for the Biomek® FX

(Click to Enlarge)
Figure 2. Diagram of the deck layout.

Figure 2. Biomek® FX initial deck configuration; 96-well purification. This is an example of a MagneSil* Total RNA mini-Isolation System deck layout on a Biomek® FX. Your specific deck layout may be different depending on your Biomek® FX configuration.

ALP Name	Equipment
Tip Loader	Biomek® P250 Barrier Tips
P1	Biomek® P250 Barrier Tips
P2	Swap position
P3	Empty
P4	Pyramid-bottom reservoir plate containing 12ml of RNA Lysis Buffer
P5	96-well, flat-bottom culture plate containing sample
P6	96-well, U-bottom plate (Binding Plate) containing 30µl of MagneSil* RNA Paramagnetic Particles per well
P7	Empty 2.2ml deep-well, 96-well plate (used for waste)
P8	96-well, U-bottom plate containing 55µl of prepared DNase Solution per well
P9	Pyramid-bottom reservoir plate containing 25ml of Nuclease-Free Water (Elution)
P10	Empty
P11	MagnaBot* 96 Magnetic Separation Device with 1/4 inch Foam Spacer
P12	Pyramid-bottom reservoir plate containing 12ml of DNase Stop Solution (ethanol added)
P13	96-well polypropylene U-bottom Collection Plate for purified total RNA (Elution Plate)
P14	Empty
P15	Pyramid-bottom reservoir plate containing 50ml of 90% ethanol

Completely resuspend the MagneSil* RNA PMPs before dispensing.

C. Initial Deck Configuration for the Biomek® FX

(Click to Enlarge)
Figure 3. Diagram of the deck layout.

Figure 3. Biomek® FX initial deck configuration; 384-well purification. This is an example of a MagneSil* Total RNA mini-Isolation System deck layout on a Biomek® FX. Your specific deck layout may be different depending on your Biomek® FX configuration.

ALP Name	Equipment
Tip Loader	Biomek® P250 Barrier Tips
P1	Biomek® P250 Barrier Tips
P2	Biomek® P250 Barrier Tips
P3	Biomek® P250 Barrier Tips
P4	Swap position (leave empty)
P5	384-well plate for purified total RNA
P6	Biomek® P250 Barrier Tips
P7	Empty 2.2ml deep-well, 96-well plate (used for waste)
P8	Pyramid-bottom reservoir plate containing 25ml of Nuclease-Free Water
P9	96-well, U-bottom plate containing 55µl of prepared DNase Solution per well
P10	Empty
P11	MagnaBot* 384 Magnetic Separation Device
P12	Pyramid-bottom reservoir plate containing 60ml of 90% ethanol
Orbital	384-well, flat-bottom cell culture plate containing sample
P13	96-well, U-bottom plate (Binding Plate) containing 50µl of MagneSil* RNA Paramagnetic Particles per well
P14	Empty
P15	Pyramid-bottom reservoir plate containing 15ml of DNase Stop Solution (ethanol added)
P16	Pyramid-bottom reservoir plate containing 20ml of RNA Lysis Buffer

Note: Use a 96-channel head for the 384-well method.

Completely resuspend the MagneSil* RNA PMPs before dispensing.

D. Description of MagneSil* Total RNA mini-Isolation Protocol

This overview describes the general liquid handling and purification steps required for RNA isolation from samples in a 96-well plate format using the MagneSil* Total RNA mini-Isolation System. This protocol can be performed manually or adapted to a variety of automated liquid handling robots. Section VII.C provides information on RNA isolation in a 384-well format. For additional information about adaptation to liquid handling robots other than those already discussed, see Section VIII.

In the protocol described below, a plate shaker (such as the DPC Micromix* 5 Shaker) is used for mixing steps. For all steps where shaking is indicated, shake the plate vigorously but not so vigorously to cause splashing/spilling, because this will cause cross-contamination of samples. Recommended shaker settings for the DPC Micromix* 5 Shaker are provided, but these may need to be optimized because settings often differ slightly between shakers.

Alternatively, mixing may be performed manually using a multichannel pipettor. Manual mixing will slow the purification process considerably. Ensure that the MagneSil* RNA PMPs are thoroughly resuspended during the purification procedure.

E. Sample Preparation

Before beginning the purification procedure, prepare the starting materials as follows:

Cultured Cells: Wash cultured cells gently once with 1X PBS. If using adherent cells, be careful not to wash the cells off the bottom of the plate. Remove all 1X PBS to waste. Purification begins with cell culture plate containing cells alone.

Tissue Lysate: Homogenize the tissue in RNA Lysis Buffer. Place up to 2mg of tissue lysate in a total volume of 100µl of Lysis Buffer per well of a 96-well plate.

Whole Blood: Place up to 20µl of whole blood into each well of a 96-well sample plate.

F. Protocol


1.	Thoroughly resuspend the MagneSil* RNA Paramagnetic Particles (PMPs) in the reagent bottle. Dispense 30µl of particles to each well of 96-well U-bottom plate. Completely resuspend the MagneSil* RNA PMPs before dispensing.
2.	Remove Storage Buffer. Move the 96-well processing plate onto the MagnaBot* Device and pause for 1 minute to capture the MagneSil* RNA PMPs to the side of the wells. Carefully remove supernatant to waste.
3.	Sample Lysis. Add 100µl RNA Lysis Buffer to each sample and mix either by tip mixing or by vigorous shaking (DPC settings: form 47, amplitude 7) for 1 minute. Note: Addition of RNA Lysis Buffer is not required for purification from tissue lysates, as these are homogenized in RNA Lysis Buffer.
4.	Capture of Nucleic Acids. Transfer the sample in RNA Lysis Buffer to each well of the 96-well U-bottom processing plate containing the MagneSil* RNA PMPs. Mix vigorously on plate shaker (DPC settings: form 47, amplitude 7) for 2 minutes.
5.	Move the 96-well processing plate onto the MagnaBot® Device and pause for 1 minute to capture MagneSil-bound nucleic acids to the sides of the wells. Remove and discard the supernatant, taking care to avoid disturbing the captured MagneSil RNA PMPs.
6.	Wash. Add 100µl of 90% ethanol to each well. Move the processing plate from the MagnaBot® Device to the shaker and shake (DPC settings: form 42, amplitude 5) vigorously for 1 minute to resuspend and wash the MagneSil* RNA PMPs. Note: For larger sample sizes (1 × 105 cells or 2mg of tissue lysate), some particle clumping may occur at Step 6. This should disperse after DNase treatment and subsequent washes.
7.	Move the 96-well processing plate onto the MagnaBot® Device and pause for 1 minute to capture MagneSil*-bound nucleic acids to the sides of the wells. Remove and discard the supernatant.
8.	Ensure that all of the ethanol wash has been removed from the wells. Incubate the plate for 1 minute on the MagnaBot® Device to allow residual ethanol to evaporate.
9.	DNase Treatment. Add 50µl of prepared DNase Solution to each well of the processing plate. Move the plate from the MagnaBot® Device to the shaker and shake to resuspend the MagneSil* RNA PMPs. Shake (DPC settings: form 47, amplitude 6) for 10?15 minutes to allow the DNase Solution to digest contaminating genomic DNA.
10.	DNase Inactivation. Add 100µl DNase Stop Solution to each well of the processing plate and shake (DPC settings: form 42, amplitude 4) for 2 minutes to resuspend the MagneSil* RNA PMPs.
11.	Move the processing plate onto the MagnaBot® Device and pause for 1 minute to capture the MagneSil*-bound nucleic acids to the sides of the wells. Remove and discard the supernatant.
12.	Wash. Add 100µl of 90% ethanol to each well. Move the processing plate from the MagnaBot® Device to the shaker and shake vigorously (DPC settings: form 42, amplitude 5) for 1 minute to resuspend and wash the MagneSil* RNA PMPs.
13.	Move the processing plate onto the MagnaBot® Device and pause for 1 minute to capture MagneSil*-bound total RNA to the sides of the wells. Remove and discard the supernatant.
14.	Repeat Steps 12 and 13 for a total of two 90% ethanol washes.
15.	After complete removal of the last 90% ethanol wash, pause for 2 minutes with the 96-well processing plate on the MagnaBot® Device to allow the MagneSil* RNA PMPs to dry.
16.	Elution. Add 50µl of Nuclease-Free Water to each well of the 96-well processing plate. Note: Elution volumes as low as 25µl can be used to increase the concentration of the eluted total RNA.
17.	Move the processing plate from the MagnaBot® Device to the shaker. Shake (DPC settings: form 47, amplitude 6) vigorously for 2 minutes to resuspend the MagneSil* RNA PMPs and elute the purified total RNA.
18.	Move the 96-well processing plate onto the MagnaBot® Device and pause for 1 minute to capture the MagneSil* RNA PMPs. Remove the supernatant containing purified total RNA to a new 96-well polypropylene plate. Store the purified total RNA at ?70°C. Note: Addition of 0.5µl of RNasin* Plus RNase Inhibitor (Cat.# N2611) to samples can help protect eluted total RNA from post-purification degradation. During elution, 0.5µl of RNasin* Plus RNase Inhibitor can be added per 50µl of Nuclease-Free Water.

G. Use of MagneSil* Total RNA mini-Isolation System in a 384-Well Format

A 96-well purification procedure has been described. 384-well purification is achieved by scaling down the 96-well purification procedure. Below is a table of relative volumes of reagents for each purification format. The purification limits for 384-well purification are: =1 × 103 cells or =5µl of whole blood. We do not recommend 384-well purification of tissue lysates.

Reagent	Volume/Well 96-well Purification	Volume/Well 384-well Purification
RNA Lysis Buffer	100µl	50µl
MagneSil* RNA PMPs	30µl	10µl
90% Ethanol Washes	100µl	50µl
DNase Solution (prepared)	50µl	12.5µl
DNase Stop Solution	100µl	25µl
Nuclease-Free Water for elution	50µl	15µl

The recommended volume for the MagneSil* Total RNA mini-Isolation System is 50µl for 96-well purification. This elution volume can be decreased to 25µl to increase the concentration of purified total RNA without a significant drop in total yield. Elution volumes less than 25µl will result in concomitant decrease in RNA yield. We do not recommend decreasing the elution volume for 384-well purifications.

H. General Guidelines for Adaptation to Alternative Robotic Platforms

The use of aerosol-resistant tips is recommended for the MagneSil* Total RNA mini-Isolation System to decrease the chance of contaminating samples with RNases. If your robotic platform uses fixed tips, be sure that the tips are washed thoroughly between pipetting steps. Also, if system liquid is used to perform pipetting steps, be sure to limit the exposure of samples to system liquid, a potential source of RNase contamination, during all pipetting steps by increasing the volume of leading air gaps that are used for pipetting.

Do not exceed 1 × 105 cultured cells, 2mg of tissue lysate, or 20µl of whole blood per well. Purification from tissue lysates can present particular problems. Do not exceed 2mg of tissue per sample well, and ensure that the sample volume is 100µl. If sample volume is less than 100µl, add Lysis Buffer to bring the volume to 100µl.

Complete resuspension of MagneSil* RNA Paramagnetic Particles (PMPs) is necessary for efficient purification of total RNA. MagneSil* RNA PMPs need to be equivalently dispensed into the sample processing plate and thoroughly resuspended during wash steps. Failure to resuspend MagneSil* RNA PMPs could result in variable yields from well-to-well, genomic DNA contamination, low yields or low purity of the purified RNA.

Addition of 0.5µl of RNasin* Plus RNase Inhibitor (Cat.# N2611) to samples can help protect eluted total RNA from post-purification degradation. During elution, 0.5µl of RNasin* Plus RNase Inhibitor can be added per 50µl of Nuclease-Free Water.

For RT-PCR analysis, RNA volumes exceeding 10% of the final reaction volume are not recommended.

* All trademarks are the property of their respective owners. Where applicable, the PCR process is covered by patents owned by Roche Molecular Systems, Inc., and F. Hoffman-LaRoche, Ltd.