DETERMINATION OF SPECIFIC ACTIVITY OF PROBE (i) Proportion of CTP incorporated into the RNA:

1.48 X 105 = 50%

2.97 X 105

(ii) Number of moles of ^^P-CTP in the reaction (calculated by converting the volume of 52p-CTP added to the number of mCi o f ^^P added, and then further converting to the molar amount using the known specific activity:

(a) 5 pi X 10 mCi/ml (5 x 10-3 x 10) = 0.05 mCi 32p-CTP (b) 0.05 mCi = 0.125 nmoles in reaction

400 X 103 mCi

(iii) Number of moles of 32p-CTP actually incorporated: 50% X 0.125 nmoles = 0.06 nmoles

100

(iv) Amount of RNA synthesised in reaction (assuming that the RNA synthesised contains equi-molar amounts of ATP, CTP, GTP and UTP) using a total molecular mass for all four ribonucleotides of approximately 1320 Da.

1320 g X 0.06 X 10-9 moles = 79.2 ng

(v) Cpm incorporated into the RNA product: The total reaction volume from which the 2 pi sample was removed to determine label incorporation was 100 pi. The TCA precipitated probe contained 1.48 x l0 5 cpm, so the whole probe contained 1.48 xlO ? TCA precipitable material.

Specific activity of the GM-CSFRa probe:

14.8 X 106 cpm = 1.78 cpm xlO^/pg

80 ng

The specific activities of the probes varied considerably (Table 3.1). It was therefore only possible to directly compare and quantify expression o f the mRNA transcripts in different experiments if the same probe had been used. In cases where different probes had been employed, it was necessary to make adjustment for the relative intensities of the probes used in each case.

GM -CSFRa GM-CSFRp Actin

Probe 1 1.78x10® 2.4x10® 0.88x10®

Probe 2 3.4x10® 4.6x10® 4.1 xIO®

Probe 3 2.1 xIO® 3.9 xIO® 1.6 xIO®

Table 3.1 The specific activities (cpm/pg) of probes synthesised in three separate experiments from a single stock of digested DNA template.

3.3.3 Sensitivity of the GM -CSFRa and p RNase protection assays

In order to establish the sensitivity of this assay and to set up a calibration curve for the quantification of receptor mRNA transcripts, the a and p antisense probes were hybridised to target synthesised RNA sense transcripts. The RNA transcripts were serially diluted to the concentration o f GM-CSFR mRNA transcripts expected in target tissue. The target sense transcripts were spiked with a constant amount of TF-1 cell total RNA, to obtain an actin signal and internally control this experiment

G M -C SFR a: The signals from the serially diluted target sense transcripts (1 pg to 1 ng) and actin are shown in Figure 3.7. The signals from the sense transcripts and actin were expressed as a ratio and a calibration curve was plotted. As the target RNA concentration decreases the calibration curve becomes non-linear (Figure 3. 8). The lower limit of detection o f the sense transcript in the GM- C SFRa mRNA protection assay was approximately 10 pg per sample, with a linear signal obtained up to 6 ng.

GM -CSFRP: The signals from the serially diluted target sense transcripts (1.25 pg to 12.5 pg) are shown in Figure 3.9. The signals from the sense transcripts and actin were expressed as a ratio and a calibration curve was plotted (Figure 3.10). As in the case of GM-CSFRa, when the target RNA concentration decreases the calibration curve is non linear.

The GM-CSFRp mRNA protection assay was more sensitive than that for G M -C S F R a , as the detection limit was approximately 1 pg per sample. However, the maximum was approximately 12 pg per sample and so the detectable range for GM-CSFRp was more restricted than GM-CSFRa. Higher amounts could not be accurately quantified above this level as the intensity of the specific GM-CSFRp mRNA signal was too high relative to the actin signal derived from the total mRNA from either HL-60 or TF-1 cells.

Amount of sen se ^ ^ transcript (pg) S 3 <d Antisense probe + sense transcript <

<

i

F igure 3 .7 RNase protection o f the serially diluted target G M -C SF R a sense transcripts containing 15 pg o f TF-1 total RN A

1000 O) Q .

<

<

F igu re 3 .8 RNase protection assay calibration curve for G M -C SF R a

Amount of se n se Antisense probe + sen se transcript Actin -► u > U ) in in csi CN N e g ( 6 CO T- < z cc -1 5 6 bp -1 3 4 bp

F igure 3 .9 RNase protection o f the serially diluted target GM -CSFRp sense transcripts containing 15 |ig o f TF-1 total RNA

12.5 2

<

The calibration curves were used to estimate the amount o f GM-CSFRa and P mRNA in 15 |ig total RNA extracted from TF-1 cells, HL-60 cells and neutrophils. The RNase protection assays were performed using the same probe in each case in order to control for differences in the specific activity. The results are summarised in Table 3.2.

It was not possible to give accurate absolute quantities on a single cell basis as it could not be assumed that the RNA recovery was similar in the different cell types. Furthermore there was considerable variation in the amount o f RNA extracted from the same cell type but carried out on different days. The variation of RNA recovery obtained using a single RNA extraction protocol is shown in Table 3.3.

HL-60 (uninduced) TF-1 Neutrophils

G M -C SFR a mRNA 1.4 4.0 1.3

GM-CSFRp mRNA 0.06 0 .0 8 0 .1 7

Table 3.2 GM-CSFa and p mRNA (pg) measured by RNase protection assay per |ig total RNA extracted from different cell types

H L -60 (u n in d u c e d ) TF-1

E x p er im e n t 1 4 .5 1 0

E x p er im e n t 2 7 0 4 0

E x p er im e n t 3 ________________ 1 2 _______________________ 8 0 _________