Spatial transcriptomics capture high-resolution spatial distributions of RNA transcripts within biological systems, yet whole-transcriptome profiling of genes at single-cell resolution remains a challenge. To address this limitation, spatial gene expression prediction methods have been developed to infer the spatial expression of unmeasured transcripts, but the quality of these predictions can vary greatly across different contexts. Poor predictions of gene expression in even a small subset of cells or genes can manifest in misleading downstream analyses. As such, there is a need for uncertainty-aware procedures for utilizing predicted spatial gene expression profiles. Here we present TISSUE (Transcript Imputation with Spatial Single-cell Uncertainty Estimation) as a general framework for estimating uncertainty for spatial gene expression predictions and providing uncertainty-aware methods for downstream inference. Leveraging conformal techniques, TISSUE provides well-calibrated prediction intervals for predicted expression values. Moreover it improves downstream analyses to consistently reduce false discovery rates for differential gene expression analysis, improve clustering and visualization of predicted spatial transcriptomics, and improve the performance of predictive models trained on imputed gene expression profiles. We have made TISSUE publicly available as a flexible wrapper method for existing spatial gene expression prediction methods to assist researchers with implementing uncertainty-aware analyses of spatial transcriptomics data.