A: Handling

1. Do I need to work in a laminar work flow?

In principle, one should use the same safety and contamination precautions as for PCR in microplates. For setting up PCR a hood can always be recommended.

2. Which pipettes are recommended to work with the AmpliGrid?

We recommend Rainin AutoRepE or Eppendorf Multipette® stream. Only electronic pipettes ensure consistent pipetting of the PCR and sealing solutions without forming satellite droplets. If only manual pipettes are available, be careful not use the second pressure point of the pipette. Otherwise, you will inject small air bubbles into the reaction mix which can cause the mix to burst during cycling. Also avoid to deposit excessive amounts of buffer when depositing cells as the buffer crystals can lead to bubble formation.

3. Why is it important to hold the pipette vertically when adding the reaction mix or the sealing solution on the AmpliGrid reaction sites?

The ideal pipetting angle is 70-80°. If the pipetting angle is lower than that, droplet deposition gets inaccurate and the risk for touching the slide surface with the pipette tip is increased. Performing a vertical pipetting results in a nice droplet formation at the end of the pipette tip and a smart deposition of the droplet onto the AmpliGrid reaction site.

4. Is it possible to run PCR on AmpliGrid with extracted nucleic acids as template instead of cells?

Yes. Please take care that your extracted nucleic acid has a good quality (like Agencourt column extraction).

5. Can I be 100% sure to amplify exactly one single cell or one single sperm on the AmpliGrid?

Yes. Based on standard microscopy methods it is possible to check the target immediately before the amplification reaction. This is one of the unique advantages of the glass slide and contrasts to any other amplification platform. For more information read the detailed description of our visual control workflow here.

6. How can single cells be deposited on the AmpliGrid?

Single cells can be deposited on the AmpliGrid using different techniques such as:
• Flow cytometry (see video)
• Micromanipulation (use 0.05x PBS to micromanipulate the cells)
• Laser microdissection (e.g., PALM system)
• Pipetting dilutions

Avoid to desposit excessive amounts of buffer when depositing cells as the buffer crystals can lead to bubble formation. Additionally large buffer amounts dramatically decrease efficiency of PCR due to changes in the buffer concentrations. This is particularly important if you hand pipette cells onto the AmpliGrid. Buffer should never exceed 0.5ul (0.05x PBS deposition buffer).

7. If I don’t use all 48 sites in my experiment, can I re-use the unused sites in a future experiment?

9. I want to perform a reaction with more than 1 µl - how much volume can I put on one reaction side?

AmpliGrid slides are designed and optimized to hold 1 μl exactly.  It is possible to increase the volume of the aqueous phase up to 2µl. Using increased volumes destabilizes the reaction mix and you may experience an elevated level of burst drops so we do not recommend it in routine.

10. Can I reuse the AmpliGrid slide?

No. The AmpliGrid slide is manufactured in a clean room production facility and is delivered certified DNA/RNA free. Once used, the reaction sites are contaminated with DNA and no longer suitable for PCR.  Additionally the hydrophobic/ hydrophilic surface characteristics are destroyed during the PCR run and drops are no longer held in place correctly.

B: Sensitivity of the system

1. How sensitive is the AmpliGrid? 

Yes, we have model systems, in which starting from a single cell the product can be visualized on a conventional agarose gel (2%). We typically see an increase in sensitivity by a factor of 3-5 (for further information please click here).

2. What is the amount of PCR product that can be achieved from a 1 µL PCR reaction?

Starting with one single cell (corresponds to 1 or 2 copies of a gene sequence) the typical outcome of a 1 µL reaction on the AmpliGrid is 1 – 100 ng of PCR product.

3. What is the amount of RT-PCR product that can be achieved from a 1 µL PCR reaction?

Starting with 1000 transcripts the typical outcome of a 1 µL reaction on the AmpliGrid is 10 – 100 ng of RT-PCR product. Please have a look here for an example of the sensitivity achieved in a single cell multiplex rt-PCR reaction.

C: Reagent-related questions

1. What is the recommended primer for reverse transcription: Oligo(dT), random hexamer or gene specific primer?

In principle the usage of all primers is possible. Higher specificity and yield of transcribed product can be achieved when using gene specific primers. Starting amounts for an optimization of the experiment on the AmpliGrid for a one µL should be:
- gene specific primer: 0.6 pmol for singleplex; 0.2 pmol for 10-plex
- Oligo(dT): 3.5 pmol
- random hexamer: 1-1.5 pmol
Example for calculating the amounts out of primer concentrations: 0.6 pmol are 0.1 µL of a 6 µM primer solution (each sense and antisense) in water.

2. Is DNase treatment recommended for single cell RT-PCR?

As the AmpliGrid slide shows an outstanding sensitivity it is generally possible to amplify single DNA copies during an RT-PCR experiment. During the analysis of high expressed genes the final amount from the DNA amplification can be neglected compared to the RNA amplification whereas analyzing medium or low expressors the amplified DNA might interfere with the results. We recommend two strategies to overcome that issue: (1) Primerdesign: Use intron spanning primers to be able to distinguish between DNA and RNA amplicons. In case you need assistance for designing the appropriate primers please make use of our service. (2) DNase treatment: if an intron spanning primerdesign is not possible please integrate a DNase step into your RT setup (see here for protocol). Please note: any additional step during a single cell experiment is prone to sample loss or degradation of RNA.

3. What is recommended for subsequent qPCR: SYBR® or TaqMan® assays?

Our workflow combines single cell RT on the AmpliGrid and subsequent quantitative analysis on any kind of qPCR cycler. Protocols for both SYBR® dyes and TaqMan® probes are available.

4. Where can I find example protocols to help me adapt my current 20µl protocol to a 1ul AmpliGrid protocol?

Please check our page here.

5. Which staining or fixation technique for cells or tissues is suitable for the AmpliGrid system?

The best results are obtained when using fresh material. We also successfully tested fixation using ethanol, methanol and paraformaldehyde (4%). Paraffin embedded material might be difficult to amplify due to the PCR inhibition properties of paraffin.

6. For what cell types do you recommend cell extraction / protease treatment?

(1) Lysis: PCR success rate is depending on the ability to crack the cells outer membrane to make the nucleic acid basically available to the PCR enzyme. This has nothing to do with the platform used but with the cell type. Some cells, especially "inner body cells" are more fragile and therefor easier to crack by temperature effects from the initial
denaturation. For hard-to-crack cells, like epithelials (buccal, some CTCs...) require an additional  chemical lysis to open the cell membrane or to make it at least kind of semi-permeable.

(2) DNA associated proteins and equivalents: Once the cell has been cracked the next obstacle is to have the sequence of interest available for attaching the primers and the polymerase. Depending on the positioning of the sequence this might not be necessary, for others this is essential. Comparing the multiplex PCR results with and w/o CEK it becomes clear that this differs from sequence to sequence. Basically the PCR works in all of the cases but the dropout rate increases in the experiments w/o CEK:
ampligrid_cell_extraction_kit_998_applications_1003.htm
Dropouts might not be fully negative but also simply not detectable in standard PCR systems. As the AG is a factor of 3-5 more sensitive compared to microplates you now get access to that data.

D: Applications

1. Can I do WGA or WTA from single cells followed by microarray analysis?

Advalytix does not currently have protocols for WGA and WTA due to the technical challenge of producing enough material in a 1ul reaction for microarray analysis (CGH or expression arrays).

2. What methodology do you recommend to study single cell molecular heterogeneity?

We suggest a hypothesis testing approach (multiplex gene specific amplification with gel, CE or qPCR analysis) instead of discovery approaches (WGA or WTA plus microarray) to study genetic heterogeneity in single cells. If it is possible to name subsets of 5-20 “genes of interest”, data is typically more reliable (bias, coverage, sensitivity), at much lower per-cell cost than data obtained from discovery methods. 

Cost: For the cost of analyzing one cell via microarray, 160 cells can be analyzed via AmpliGrid based multiplex PCR.  ($5 per cell for multiplex gene-specific PCR with AmpliGrid workflow, including cell deposition via flow cytometry and gel or CE analysis vs. $800 for WGA/ WTA and microarray analysis)
Bias: Particularly for the small amounts of DNA or RNA in single cells, WGA/ WTA can introduce an amplification bias of 10³ to 10⁶ (Xu et al., 1993). The high single cell PCR efficiency on the AmpliGrid avoids systematic underreporting of low-abundance genes.
Coverage: In WGA/ WTA, restriction of template prior to amplification results in incomplete genome coverage and some percent of sequences are lost. In comparison, gene-specific multiplex PCR can be designed to guarantee amplification of all target genes.
Sensitivity: The absolute sensitivity for amplifying a specific gene is higher for gene-specific assays than for WTA/ WGA due to the higher concentration of gene specific primers and absence of competition from random primers. 

3. Can I do single cell methylation analysis?

No we do not yet have a single cell methylation protocol, but for a methylation protocol involving cell incubation, look here.