2.2 SISTEMA DE IMPRESIÓN DE TICKETS (SIT)
2.3.3 REPORTES DE TICKET
None o f the conditions for the reverse transcription reaction were altered at any point. This was therefore routinely carried out as outlined in the Methods, section 2.3.8.
3.10.3 PCR
cDNA in different DDRT-PCR reactions. 1/3 0th, 1/15th, 2/15th and 4/15th o f the total reverse transcription reaction (which was usually a total volume of 3 Dpi) were compared in separate PCR reactions with no discernible difference seen between the reactions (results not shown). 1/30th o f the reverse transcription reaction was therefore used in subsequent experiments as this maximises the number of PCR reactions that can be performed from the cDNA produced by a single reverse transcription reaction.
Varying amounts of d-ATP were used in the PCR reaction: 6.25pCi (0.5pi), 3.75pCi (0.3pl) and 3.125pCi (0.25pl). 0.3pl of d-ATP in a lOpl PCR reaction volume was found to be the minimum amount of radionucleotide required to produce sharp bands after an overnight exposure o f the acrylamide gel to X-ray film (results not shown).
Two brands o f Taq polymerase enzyme were tested in the radioactive PCR reaction: Promega and Advanced Biotechnologies. No products were seen with the Red Hot Taq polymerase from Advanced Biotechnologies (results not shown), whilst Promega Taq showed consistent amplification of products; this enzyme was therefore used throughout. 0.5 units, 1 unit and 2 units of enzyme were tested and 1 unit was found to produce the best PCR results (figure 3.6, results not shown for 2 units, but results were similar to 1 unit).
An experiment was carried out where the annealing temperature o f the PCR reaction was varied. No PCR products were seen when using an annealing temperature of 40°C was used, and no products or only a few with 41°C (figures 3.7 and 3.8). An annealing temperature of 42°C was therefore used in all subsequent reactions. This result was not expected; usually when a lower annealing temperature is used there are a greater number of non-specific amplification products produced. There is no obvious explanation for this result.
0.5 units 1 unit Taq Taq (Q •D E (0 230bp 142bp
i
Figure 3.6 Varying the amount o f Promega Taq polymerase enzyme used in DDRT- PCR. Gel shows mouse testis DDRT-PCR products electrophoresed for 1 hour at SOW (VA anchor, OP-1 10-mer primers). 0.5 units enzyme is compared with 1 unit o f enzyme. Marker ladder is lambda DNA digested with Hinfl; size is noted in base pairs
40°C annealing temp. 42°C annealing temp. c
i i l l I
O)c D (D E .<2 E % (Ü .0) (D - c.1 s
^ I Ç 2 0Û O) c 3 230bp8
Figure 3.7 Varying the annealing temperatures for DDRT-PCR. Gel shows mouse testis, liver, kidney, heart, brain and lung DDRT-PCR products electrophoresed for 1 hour at SOW (VA anchor, OP-1 10-mer primers). Comparison is between annealing temperatures o f 40°C and 42 XI. Marker ladder is lambda DNA digested with Hinfl; size is noted in base pairs to the left o f the ladder.
42°C annealing temp. 41 °C annealing temp.
I I 11 II
I I
tr(0 <D I 0) c ■g k11
Figure 3.8 Varying the annealing temperature for DDRT-PCR. Gel shofws mouse testis, liver, kidney, heart, brain and lung DDRT-PCR products electrophoresed for 1 hour at 30fV (VA anchor, OP-3 10-mer primers). Comparison is between annealing
The magnesium concentration within the PCR reaction was also altered to see whether this could be optimized. Magnesium concentrations of ImM, l.SmM, 3mM, 3.5mM, 4mM and 4.5mM were tested. At an annealing temperature of 42°C, magnesium concentration appeared to have little effect on the PCR reaction; figure 3.9 shows Mg^^ concentrations of 3mM, 3.5mM, 4mM and 4.5mM compared for mouse testis and liver DDRT-PCR products. A similar Differential Display pattern can be seen in all reactions. A Mg^^ concentration of 1.5mM was therefore used routinely as the Promega Taq
polymerase enzyme buffer already contained magnesium at this concentration.
The PCR reaction was first carried out in a total reaction volume of 20pl but this was later reduced to lOpl as only 1/lOth of the PCR reaction was electrophoresed through the polyacrylamide gel. Varying amounts of the PCR reaction were loaded onto the gels and there appeared to be little effect on the band intensity seen after exposure to X-ray film. Routinely, l-2pl fi-om a lOpl PCR reaction was loaded onto a polyacylamide gel for electrophoresis.
3mM 3.5mM 4mM 4.5mM
T L T L T L T L
t
t
KEY; T = Testis L = Liver
Figure 3.9 Varying magnesium concentrations in DDRT-PCR. Gel shows mouse testis and liver DDRT-PCR products electrophoresed for 1 hour at SOW (VA anchor, OP-1 10-mer primers). Comparison is o f magnesium concentrations between 3 -4.5 mM at an
3.10.4 Polyacrylamide gel electrophoresis
After electrophoresis, sharper bands were seen after exposure to X-ray film when the PCR products were loaded onto non-denaturing as opposed to denaturing (urea- containing) gels. Non-denaturing gels are easier and quicker to prepare than denaturing gels and so were used preferentially.
Varying concentrations of polyacylamide gel were tested: 4%, 6% and 8%. The best separation of PCR products was seen with the 6% gel with no loss of the number of bands that could be visualized in a single run on a 4% gel. Products were smeared when electrophoresed through an 8% gel.
A sharkstooth comb such as that used for sequencing gels was preferential to the standard comb used for fingerprinting as the slightly wider wells meant that for the amount of PCR product that was loaded on the gel, the bands were sharper and more definite.
The non-denaturing polyacrylamide gel was usually electrophoresed for between 50 minutes and an hour. This produced a separation of PCR products that could clearly be distinguished from one another ranging in size from 20 base pairs to over 300 base pairs. Although there appeared to be products greater in size than this, these could not usually be separated even when the gel was electrophoresed for an extra 2 hours.
To determine the size the DDRT-PCR products, a radioactive size marker (bacteriophage Lambda DNA digested with HinQ. enzyme and end-labelled with a^^S- dATP) was electrophoresed adjacent to the samples on each acrylamide gel. For products that were electrophoresed through denaturing polyacrylamide gels, a radioactive ladder was produced by sequencing single-stranded bacterial phage Ml 3 with dideoxy-guanosine chain terminator in the presence of a^^S-dATP. The sizes of each of the bands produced by both of these methods were known and both proved to be adequate size markers.
The radioactive DDRT-PCR products were loaded onto the non-denaturing polyacylamide gel with a formamide loading dye which helps to separate the double stranded PCR products. It has been suggested that samples should also be denatured at 80°C for 2 minutes before loading (Reeves et a l, 1995). I found this produced no discernible difference in the quality or number of cDNA bands and so I omitted it from the standard protocol.
3.10.5 Re-amplification of excised cDNA fragments by PCR
After excising a cDNA band of interest fi*om the gel after the gel had been removed onto Whatman 3MM paper and exposed to X-ray film, the DNA was eluted in lOOpl of TE buffer. At this stage the gel slice was often still covered with cling film which did not seem to impede the elution of the DNA from the Whatman paper into the buffer.
After elution of the DNA, a fraction of the eluted sample was re-amplified by PCR. This reaction did not work at an annealing temperature of 42°C (several attempts were made) and so the temperature was reduced to 40°C instead. At this temperature a single PCR product corresponding in size to the band excised from the polyacrylamide gel was usually seen.
Promega Taq polymerase was again found to be most satisfactory for the re amplification of the cDNA fragments, although Biotaq (Bioline) also worked well, and indeed amplified DNA from some eluants that could not be amplified by Promega Taq.
In my hands, this stage of DDRT-PCR was the most unreliable and it was difficult to pinpoint exactly how to improve the efficiency of this step. The DNA eluted from excised fragments only re-amplified by PCR in about 65% of the cases (see tables 3.1 and 3.6). Several alterations of the standard PCR conditions were made, but no improvements in the re-amplification success rate were achieved. It was thought that the variable amounts of cDNA present in the excised fragments may not always be amplified by the standard PCR reaction, and so for many of the cDNAs that failed to re-amplify, I carried out a number of separate PCR reactions using differing amounts of eluted DNA as a substrate (0.5 pi, Ipl, 2pl, 3 pi, 5 pi and lOpl of the eluted DNA). Usually, Ipl or 2pl of the eluted product was sufficient for re-amplification and if these amounts did not amplify, it was only on a very rare occasion that a different amount did. Figure 3.10 shows the re-amplification of DDRT cDNAs llVGa, llVGb and llVGc; the DDRT-
PCR gel from which these fragments were excised can be seen in figure 3 .f. 1,2 and 3 pi of eluted DNA were amplified for each cDNA and different amplification results were seen for all three. All three elution amounts amplified the correct sized products for
11 VGa and 11 VGb; but for 11 VGc, only Ipl of eluted DNA was able to amplify a single product of the correct size. No correlation could ever be made with the intensity of the radioactive band in the DDRT-PCR gel and the amount of eluted DNA required to
reamplify the cDNA; in this case the bands seen in the radioactive gel (see figure 3.5) were of equal intensity.
The cycling conditions of the PCR reaction were also altered to: denaturing at 94°C for 40 seconds (fi"om 30 seconds), annealing at 40°C for 2 minutes (from 1 minute)
and extension of the products at 72°C for 40 seconds (from 30 seconds) with no effect (results not shown).
It was also thought that the re-amplification of the small cDNA fi-agments may have been impeded by there being too many cycles in the PCR reaction. To see whether this would affect the re-amplification of cDNAs, a control experiment was set up whereby a DDRT-PCR cDNA that was known to re-amplify successfijlly was amplified using varying number of cycles of the PCR reaction. 15, 20, 25, 35 and 40 cycles were tested and the optimum amplification of the cDNA was achieved with 40 cycles (figure 3.11).
The only change to the PCR re-amplification conditions that was found to produce an effect was carrying out the reaction in a total volume of 20pl as opposed to 50pl and so this was carried out routinely for all products. This suggested that it was the relative amount of eluant in the PCR reaction that was affecting re-amplification.
No other components or quantities were varied in the DDRT-PCR reactions.
11 VGa IIVGb lOObp ^[^\ 2pl 3pl ^\i\ 2[i\ 3pl ladder -llO b p 230bp 100bp Ipl 2pl 3pl ladder 11 VGc
Figure 3.10 Re-amplification by PCR of human DDRT cDNAs 11 VGa, IIVGb and llVGc, specific to testis, excised from acrylamide gel shown in figure 3.4. Ipl, 2pl and 3 pi of eluted DDRTfragment was amplified in a 20 pi PCR reaction volume and lOpl of this was electrophoresed through a 2% agarose gel for 30 minutes. The DDRT-PCR product did not amplify when 2 or 3 pi of the elutedfragment for 11 VGc was used in the
15 20 25 35 40 cycles
~140bp
i
Figure 3.11. Varying the number of amplification cycles in the PCR re-amplification reaction. PCR re-amplification of human DDRT cDNA 13VA1.
3.11 Differential Display of RNA isolated from adult male mouse tissues
Initially, Differential Display RT-PCR was carried out on RNA isolated from mouse tissues. These RNAs were used whilst I was establishing the appropriate conditions for DDRT-PCR as the tissues were plentiful, and the complete panel of human tissues was not available to me at the time. I was however, most interested in eventually isolating human testis-specific cDNAs, and so as soon as the DDRT-PCR conditions were established as routine, the experiments using mouse RNA were discontinued. To begin with, the technique was tested on RNA isolated from just mouse testis and liver. After the technique was proven to work satisfactorily, RNA isolated from mouse kidney, heart, brain and lung were included in the display reactions.
3.11.1 Negative controls for reverse transcription reactions
A negative control was carried out for the mouse liver RNA to ensure there was no DNA present in the sample that could affect the PCR results. A comparison was made before the RNA had been treated with DNAse enzyme and after; before DNAse treatment there were a few amplification products, but after DNAse treatment these few bands were much less intense (figure 3.12). The other mouse tissue RNAs were treated with DNAse enzyme in the same manner.
0: 1 1 ■O
I
z
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<
311 bp 230bp 142bpFigure 3.12 No reverse transcription enzyme control for mouse liver RNA. RNA is compared before and after treatment with DNAse enzyme. Gel shows mouse testis DDRT-PCR products electrophoresed for 1 hour at SOfV (VA anchor, OP3 10-mer primers). Marker ladder is lambda DNA digested with Hinfl; size is noted in base pairs
to the left o f the ladder.