LA ENSEÑANZA DE LA LITERATURA EN EL CENTRO ESCOLAR GUSTAVO

quantified by measuring the signal derived after PCR amplification of its

corresponding cDNA. Two assumptions are thus made:

i. that the amount of DNA in the PCR amplicon is reflected by the signal measured, with the implication that the detection system must not be limiting, and

ii. that the specific cytokine mRNA in the cells is in direct proportion to the quantity of PCR amplicon generated, by a factor related to the efficiency of the reverse-

transcription and PCR processes.

The following experiments were carried out to test these assumptions on the experimental system designed.

2.5.1 A m p lico n detection

Preliminary experiments established that the detection system used was sensitive enough to distinguish two-fold differences in the quantity of PCR amplicons. Duplicate samples loaded in different wells yielded very similar results and fluorescence intensity correlated linearly with amplicon quantity (Figure 3-6).

4 0 0 0 0 3 0 0 0 0 - 2 0 0 0 0- 1 0 0 0 0 - 1st reading 2nd reading

T

PCR amplicon 2-fold dilutions

F ig u re 3 -6 . A m p lic o n d e te c tio n by gel e le c tr o p h o r e sis. D o u b lin g d ilu tio n s o f th e sa m e a m p lic o n w ere q u a n tifie d b y e le c tr o p h o r e sis. F lu o r e sc e n c e in te n sity o f e a ch band w a s lin ea rly c o rrela te d w ith a m p lico n q u a n tity , w h ic h d e m o n stra te s that the d e te c tio n sy s te m w a s a b le to d istin g u is h tw o - f o ld d iffe r e n c e s in a m p lic o n q u antity. T h e figu re sh o w s o n e rep resen tative ex p e rim en t o f six p erform ed in d u p lica te , and the c o rr esp o n d in g g el im a g e o f all the a m p lico n s.

2 . 5 . 2 T i t r a ti o n a n a l y s i s : P C R a m p l i c o n s c o r r e l a t e w ith i n i t i a l R N A c o p y n u m b e r

Samples containing 10 -10^ copies of EL-4 cRNA standards were amplified using 15 cycles for Rl and 25 cycles for R2 as determined previously. The standard curve relating RNA copy number to amplified products was linear, confirming that the reaction conditions for RT-PCR did not cause saturation of the nested PCR reaction, within the limits of this range of starting material (Figure 3-7).

A.

_B.

IL-4 oRNA copy number

— 200

# # — 1 00 1 6 5

F ig u r e 3 -7 . c R N A standard cu rve. T he full range o f IL -4 c R N A stan dards (1 0 - 10^) w a s rev erse- transcrib ed and the c D N A a m p lifie d u sin g n ested P C R (1 5 c y c le s R l , 25 c y c le s R 2 ). T h e a m p lico n s w e re q u a n tified as d e scrib ed p re v io u sly and the resu lts p lotted a gain st the initial nu m ber o f c o p ie s o f c R N A u sed . T h e figu re s h o w s on e rep resen tative ex p e rim en t out o f ten perform ed in d ep ed en tly .

A : E a ch data p oin t rep resen ts the m ean and S E M o f trip licate a ssa y s. W h ere error bars are not se e n , th e error v a lu e s fall w ith in the sy m b o ls . B; T h e c o rr e s p o n d in g g e l im a g e o f th e IL -4 a m p lic o n s d e r iv e d from 10 - 10^ c R N A c o p ie s is sh o w n in la n es 1 - 6 r e s p e c tiv e ly , a lo n g s id e a 1 0 0 -b p D N A ladder.

2.5.2 Equivalent efficiency o f amplification o f standard and target The cDNA sample from one donor and one appropriate dilution of the standard were subjected to kinetic analysis by examining the accumulation of their amplicons over different numbers of PCR amplification cycles. When their amplicons were quantified and plotted against cycle number, the target and standard curves had similar gradients (Figure 3-8). This indicates that the standard and target had equivalent amplification efficiencies within this range of cycle numbers.

T a r g e t

S t a n d a r d

T a r g e t

- •- •'O ---- Standard

1 8 2 0 2 2 2 4 2 6 2 8

F ig u re 3 -8 . A m p lific a tio n e f fic ie n c y o f target a n d sta n d a r d . A c D N A s a m p le fro m o n e d o n o r w a s u s ed a s the te m p la te for n e ste d PC R . In d iffe r e n t rea c tio n s, 2 0 , 2 2 , 2 4 or 2 6 c y c l e s o f R 2 w e r e u s ed . T h e ta r g e t and standard c D N A ( c D N A s y n th e s iz e d from 10^ c o p i e s o f I L - 4 c R N A s t a n d a r d ) w e r e a m p lifie d in d iffe r e n t tu b es w ith in th e sa m e ex p e r im e n t, u sin g the sa m e P C R rea ctio n m ix an d c o n d it io n s . T h e ta r g e t and sta n d a r d a m p lic o n s w e r e q u a n tifie d a n d th e r e s u lts plotted a g a in st c y c le num ber. T h e gra d ien ts o f th e tw o c u r v e s are a lm o s t id e n tic a l, w h ic h d e m o n s tr a te s th at th e sta n d a rd an d target re a c tio n s p r o c e e d e d w ith e q u iv a le n t le v e ls o f e f fic ie n c y . O n e r ep r e se n ta tiv e e x p e r im e n t o f th r e e is s h o w n , t o g e t h e r w i t h th e c o rresp o n d in g g e l im a g e o f the a m p lico n s.