Product/Service

Pinpoint Slide RNA Isolation System I™

Source: Zymo Research
The Pinpoint Slide RNA Isolation System I™ is an innovative product designed by Zymo Research to isolate RNA in any targeted microscopic tissue area on a slide.
Highlights
  • Easy isolation of total RNA from microscopic tissue areas on a slide.
  • Simple procedure combines Pinpoint tissue sampling technology with a one-step RNA extraction/purification method.
  • Ultra-pure RNA for reliable RT-PCR analysis.

General Description
The Pinpoint Slide RNA Isolation System I™ is an innovative product designed by Zymo Research to isolate RNA in any targeted microscopic tissue area on a slide. The system combines our powerful Pinpoint tissue sampling technology with a one-step RNA extraction method followed by quick Zymo-Spin Column purification. This system makes targeted tissue RNA isolation simple and quick. The kit efficiently recovers RNA from fresh tissue sections for RT-PCR analysis. There is no phenol, chloroform, or other toxic reagents used in this method. There is also no need for expensive specialized equipment or computer software.
Method Principle: simply apply the Pinpoint Solution to a selected microscopic tissue area. The solution will air-dry into a thin film while the underlying cells will become embedded within. The area is lifted and transferred into a tube. After treatment with RNA Extraction Buffer, the RNA is easily recovered in 8 ul by our specially designed Zymo-Spin Column. RNA is ready for RT-PCR analysis.

Protocol
Pinpoint Slide RNA Isolation System I works best with fresh or frozen tissue sections that are fixed by ethanol, acetone, methanol, etc. Although RNA can also be recovered from old archives and paraffin embedded tissue, we found that recovery of RNA from old archives, formalin fixed, paraffin embedded tissue, was much less efficient than from fresh tissue sections. This was especially the case for amplification of relatively large fragments of cDNA over 500 bp and low abundant mRNA.

The whole procedure includes 3 parts: 1) Preparation of Tissue Sections, 2) Pinpoint fractionation to recover tissue from slide, and 3) RNA Extraction for total RNA recovery. All reagents used should be RNase free.

Before starting: Add 16 ml of 100% ethanol to the RNA Wash Buffer Concentrate.

Section 1: Preparation of Tissue Sections

    1. Rinse the glass slides several times in ethanol and sterile water, and then autoclaved or baked at 300°C for 4 hours.
    2. Mount a tissue section with >10 um thickness onto a glass slide and dry it at 60°C for 30 minutes.
    3. Drop the slide in 95% ethanol at room temperature for 60 minutes to fix the section.
    4. Air-dry the slide for about 30 minutes. Now it is ready for RNA isolation using the Pinpoint Slide RNA Isolation System.

Section 2: Pinpoint Fractionation
The purpose of this procedure is to remove the selected tissue area from the slide.

    1. Apply the Pinpoint Solution over the area where the RNA is to be recovered on the prepared slide.
    Use a sterile pipette tip or a syringe to gently spread a small amount of Pinpoint Solution over the selected region. Use about 0.5 ul of Pinpoint Solution per mm² of tissue area or cover the selected area with a layer ~0.5 mm thickness of Pinpoint Solution. It is OK to spread the Pinpoint Solution beyond the area that you had selected. It is actually easier to define the exact tissue area for recovery in step 3, see below.
    2. Dry the Pinpoint Solution completely at room temperature. It usually takes about 30 to 45 minutes.
    The Pinpoint Solution will form a thin blue film after completely drying; the underlying tissue cells are embedded within the film at this stage.
    3. Remove the embedded tissue from the slide.
    Use a sterile scalpel or sharp blade to cut the exact area you want, and then peel the area off the slide. Transfer the film to a 1.5 ml tube. Generally, a minimal 1 mm² fresh tissue area with 10 um thickness (about 500-1000 cells depending on different tissue and cell density) is needed for successful cDNA amplification. The size of each sample spot can vary from 1 to over 100 mm² according to your needs.
    4. Centrifuge the tubes briefly to bring the fragment to the bottom of the tube.

Section 3: RNA Extraction
This procedure will extract and purify total RNA from the recovered tissue.

    1. Add 200 ul of RNA Extraction Buffer to the tube containing the recovered tissue.
    2. Pipette RNA Extraction Buffer up and down to completely lyse the transferred blue film. Vortex briefly.
    3. Incubate the tube on ice for 30 minutes. Vortex tube about every 10 minutes.
    4. Add 200 ul of 100% ethanol to the tube. Mix. Incubate the tube on ice for 10 minutes.
    5. Transfer the mixture to a Zymo-Spin Column and place the column into a 2 ml Collection Tube.
    6. Spin the column at full speed in a microcentrifuge for 1 minute. Empty the flow-through.
    7. Add 200 ul of RNA Wash Buffer to the Zymo-Spin Column and centrifuge at full speed for 1 minute.Add another 200 ul RNA Wash Buffer and repeat the centrifuge step above.
    8. Transfer the column to a new 1.5 ml tube.
    9. Add 8 ml of RNA Elution Buffer directly to the membrane of the Zymo-Spin Column. Wait for 1 minute. Spin briefly to elute the RNA.

The eluted RNA can now be used for RT-PCR amplification or can be stored at -70°C for future use. Use 2-8 ul of the purified RNA for each RT-PCR reaction in a 20-50 ul reaction volume.

Troubleshooting
Following are the most common reasons for no RT-PCR amplification.

    1.RNA degradation
    RNA is very easily degraded in many situations. We emphasize the use of fresh tissue slides. If the slide cannot be processed immediately, store the slide at or below -70°C or submerge the slide in 95% ethanol at -20°C. Long term storage of tissue sections for over a month at room temperature, especially the old paraffin embedded tissue, is not recommended. If the recovered RNA is not used immediately it is recommended to add 1 unit of RNase inhibitor to the eluted RNA and store the sample at -70°C for future use.
    2. Not enough RNA to amplify.
    Make sure you choose the appropriate sample area in the beginning. Select a tissue sample area that contains more than 50 cells; increase the sampling size if dealing with sections containing fewer cells such as fat tissue and connective tissue. Tissue sampling size can be from 1 mm² to over 100 mm², depending on your particular situation. We recommend that the thickness of tissue sample is >10 nm.
    3. RT-PCR parameters are not optimized.
    The optimization of RT-PCR parameters depends upon the cDNA to be amplified and PCR conditions. We recommend that you optimize your RT-PCR condition with control RNA samples at different concentrations first, for example, 0.1 ug, 0.25 ug, and 0.5 ug, for the first set-up. Then use the best conditions for your RNA sample from the tissue slide. An increase in annealing and extension time and the number of RT-PCR cycles are recommended for low abundance mRNA. Use a more sensitive method, such as 32P or fluorescence labeling when the RT-PCR product is not evident on agarose gel stained with ethidium bromide.
    4. DNA contamination
    Generally, traces of fragmented DNA are always present in all methods for total RNA isolation from cells. To increase the specificity of RT-PCR, you can design the primers annealing to sequences in exons on both sides of an intron or have one primer annealing on an exon/exon boundary of the mRNA. It is helpful to have a control experiment in which no reverse transcriptase is used prior to PCR. Under some specific situations, you can also treat the eluted RNA with DNase I to remove the DNA.

Zymo Research, 625 West Katella Ave, Suite 32, Orange, CA 92867-4619. Tel: 714-288-9682; Fax: 714-288-9643.