The RNA transcript levels of all human leukocyte antigen class II loci were determined from class II congenital immunodeficient B cells by polymerase chain reaction amplification of cDNA. No mRNA was observed under conditions in which 0.01% normal levels could be visualized. Pre-mRNA could be amplified from normal B cells but not from immunodeficient B cells, indicating a transcription defect.Class II A number of cis elements have been described in the regions 5' of the class II loci. These elements bind factors that are responsible for the regulation of expression of these loci (5,9,10,17,19,21,(26)(27)(28). Transgenic mice in which one or more of these elements have been deleted show abnormal tissue expression of class II products (29).In humans, regulatory mutants have been induced in cultured B-cell lines (1,4,11) (Table 1). The two primers always spanned at least one intron-exon boundary. Only the normal B-cell RNA lanes showed a cDNA amplification band of the expected size (Fig. 1), although the CID actin mRNA control was normal. Some primer combinations generated bands other than those expected. These bands often appeared stronger in the CID lanes and represent alternate cDNA primings-amplifications, which are enhanced when normal templates are not available. A faint band in the DRa CID sample migrated close to the position expected for the actual transcript. Use of another pair of primers and blot hybridizing with a DRa probe showed that this band was not DRa (data not shown).The limit of resolution of the PCR technique was determined by mixing experiments wherein normal B-cell RNA was diluted with CID RNA. Amplified HLA-DR cDNA bands were still visible (Fig. 2) in the 1/10,000 dilution, indicating that even 0.01% of normal mRNA levels in the CID cells would have been detected.Analysis of pre-mRNA by using PCR. Initial protocols used primers proximal to the promoter region, with the cDNA synthesis primer in the first intron and the amplification primer in the signal sequence. This would detect premRNAs that had elongated into the first intron. However, this protocol also amplifies any DNA contamination in the RNA preparation, since controls in which the reverse transcriptase is left out of the reaction mixture often show bands almost equal in intensity to those reactions that are reverse transcribed.The polyadenylation of pre-mRNA was used to preferentially amplify RNA and not DNA contamination. One primer consisted of the last six bases of the 3' untranslated region of the pre-mRNA and oligo(dT). The other primer was in the last intron in the pre-mRNA. The presence of amplified DNA bands of the correct size would indicate the presence of pre-mRNAs that had undergone 3'-end maturation but had not yet spliced out the last intron. Pre-mRNA was converted to cDNA by using oligo(dT) and reverse transcriptase. Using primers specific for DR transcripts, the pre-cDNA was amplified from normal B cells (Fig. 3,