Table 5 Troubleshooting suggestions

3

Inconsistent staining in the positive control sample (as in Figure 4A and B)

i) RNA is degraded

i) Reduce the time between harvesting plant tissue and placing it in fixative. Follow general rules for working with RNA to prevent contamination with RNases (see also start of protocol).

ii) Over-fixation

ii) Reduce overall fixation time, but increase pressure/time of vacuum infiltration to ensure fixative penetrates.

iii) Under-fixation (tissue may be too large for fixative to penetrate).

iii) Pieces must be kept at a maximum of 5x5 mm to ensure fixative can penetrate entire tissue. Increase pressure/time of vacuum infiltration to ensure fixative penetrates.

11, 20

Poor morphology (as in Figure 4C)

i) Long axis of sample is not perpendicular to blade on vibratome

i) Be quick when orienting the tissue pieces in the molten agarose and place the sample on ice for the agarose to solidify. Examine the first few sections cut on the vibratome and if they appear smeared, adjust the orientation of the block by either tilting the adjustable stage or removing the agarose block and making sure the side stuck on the stage is perfectly flat.

ii) Sections have been damaged during processing

ii) Be careful not to damage the tissue sections during processing (use a paintbrush for manipulation, do not vortex or centrifuge tubes, prevent pipette tip from contacting sections during multiple rounds of pipetting, consider performing experiments on-slide).

29

Nuclear staining throughout the sample (as in Figure 4D)

Amplification of gDNA

Design primers that are split across an exon-exon junction to eliminate the possibility of amplifying gDNA. Alternatively, design primers that have 1 or more large introns between them such that reducing the elongation time during PCR only allows amplification of the smaller cDNA product.

Increase the incubation time of the DNase treatment or change the DNase enzyme.

45, 60

Staining of sections appears very dark

Saturated staining

Reduce the number of cycles in the PCR and/or reduce the overall staining time.

60

Positive result in the negative “no RT” control or weak non-specific staining in the positive control

Background staining caused by presence of endogenous alkaline phosphatase enzymes

Add levamisole to the substrate to inhibit endogenous alkaline phosphatases.